Corynebacterium glutamicum genes encoding phosphoenolpyruvate:sugar phosphotransferase system protei

专利摘要:
Lt; RTI ID = 0.0 > PTS < / RTI > nucleic acid molecule and encodes a novel PTS protein from Corynebacterium. The present invention also provides an antisense nucleic acid molecule, a recombinant expression vector containing the PTS nucleic acid molecule, and a host cell into which the expression vector is introduced. Further, the present invention provides isolated PTS proteins, mutant PTS proteins, fusion proteins, antigenic peptides and seeds. Based on genetic engineering of the PTS gene in glutamicum. Lt; RTI ID = 0.0 > glutamicum. ≪ / RTI >
公开号:KR20020025099A
申请号:KR1020017016970
申请日:2000-06-27
公开日:2002-04-03
发明作者:마르쿠스 폼페유스；부르크하르트 크뢰거；하르트비히 슈뢰더；오스카 젤더；그레고르 하베르하우어
申请人:스타르크, 카르크；바스프 악티엔게젤샤프트；
IPC主号:

专利说明:

PHOSPHOENOL PYRUVATE: CORNEBACTERIUM GLUTAMICUM GENES ENCODING PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM PROTEINS Coding for the sugar phosphotransferase system protein
[1] Related application
[2] This application claims priority to U.S. Provisional Patent Application Serial No. 60 / 142,691, filed July 1, 1999, and U.S. Provisional Patent Application No. 60 / 150,310, filed August 23, 1999, , The entire contents of which are incorporated herein by reference. The present application also claims priority to German Patent Application No. 19942095.5 (filed 3 September 1999) and German Patent Application No. 19942097.1 (filed September 3, 1999), the entire contents of which are incorporated herein by reference Which are incorporated herein by reference.
[3] Some products and by-products of metabolic processes that occur spontaneously in cells are used in a variety of industries, including the food, feed, cosmetics and pharmaceutical industries. These molecules, collectively referred to as "fine chemicals", include organic acids, proteinaceous and nonproteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatics, vitamins and cofactors, and enzymes. Their production is most conveniently accomplished through the large-scale cultivation of bacteria that have been developed to mass produce and secrete the desired specific molecules. An organism particularly useful for this purpose is Corynebacterium glutamicum, a gram-positive non-pathogenic bacterium. Through strain selection, a number of mutant strains have been developed that produce a range of desirable compounds. However, the selection of improved strains for the production of specific molecules is a time consuming and difficult process.
[4] SUMMARY OF THE INVENTION [
[5] The present invention provides novel bacterial nucleic acid molecules having a variety of uses. These applications include identification of microorganisms that can be used in the production of fine chemicals as reference points for mapping genomes of Corynebacterium glutamicum and as markers for transformation; Regulation of production of fine chemicals in Corynebacterium glutamicum or related bacteria; And typing or identification of Corynebacterium glutamicum or related bacteria. These novel nucleic acid molecules herein encode proteins called phosphoenolpyruvate: sugar phosphotransferase system (PTS) proteins.
[6] Corynebacterium glutamicum is a gram-positive aerobic bacterium commonly used industrially for the mass production of a variety of fine chemicals, the degradation of hydrocarbons (for example, mineral spirits) and the oxidation of terpenoids. Thus, the PTS nucleic acid molecules of the present invention can be used to identify microorganisms that can be used, for example, to produce fine chemicals by fermentation processes. The expression of the PTS nucleic acids of the present invention or the PTS nucleic acid molecules of the invention Can be used to regulate the production of one or more fine chemicals from microorganisms (for example, to improve the yield or production of one or more fine chemicals from Corynebacterium or Brevibacterium species) ).
[7] The PTS nucleic acid of the present invention can be used to identify Corynebacterium glutamicum or an organism that may be a microorganism closely related thereto or to detect the presence of Corynebacterium glutamicum or a microorganism closely related thereto in a mixed microorganism population It can also be used to confirm. The present invention relates to a method of probing an extracted genomic DNA in a culture of the same or mixed microbial population with a probe spanning the region of the Corynebacterium glutamicum gene present only in Corynebacterium glutamicum under stringent conditions, Lt; RTI ID = 0.0 > Corynebacterium glutamicum < / RTI > gene. Although Corynebacterium glutamicum itself is not pathogenic, it is associated with adult adulthood in humans, such as Corynebacterium diphtheria (a pathogen of diphtheria), and the detection of such organisms is clinically significant.
[8] The PTS nucleic acid molecule of the present invention may also serve as a reference point for mapping the genome of Corynebacterium glutamicum or the genome of related organisms. Similarly, these molecules, variants or portions thereof, may act as markers for genetically engineered Corynebacterium or Brevibacterium species.
[9] The PTS protein encoded by the novel nucleic acid molecule of the present invention is, for example, May carry high energy carbon-containing molecules such as glucose in glutamicum, or may be involved in intracellular signaling in the microorganism. The availability of cloning vectors for use in Corynebacterium glutamicum, such as the cloning vectors disclosed in U.S. Patent No. 4,649,119 (Sinskey et al.); And genetic engineering techniques of Corynebacterium glutamicum and related Brevibacterium species (e.g., Lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J Given that the nucleic acid molecule of the present invention is a more efficient producer of one or more fine chemicals, it is preferred that the nucleic acid molecule of the present invention be a < RTI ID = 0.0 > , ≪ / RTI > which can be used to genetically manipulate the organism.
[10] The PTS molecules of the present invention can be modified to improve production yield, yield and / or production efficiency for one or more fine chemicals. For example, by modifying the PTS protein involved in glucose uptake to be optimized in the active aspect, the glucose uptake or the rate at which glucose moves into the cell can be increased. The energy that can be used to cause a biochemical reaction, e.g., a reaction involving the biosynthesis of a fine chemical substance, which is difficult to occur energetically due to the degradation of glucose and other sugars in the cell is obtained. This degradation also provides intermediates and precursor molecules necessary for biosynthesis of certain fine chemicals such as amino acids, vitamins and cofactors. By modifying the PTS molecule of the present invention to increase the amount of intracellular high energy carbon molecules, the energy available to carry out the metabolic pathways required to produce one or more of the fine chemicals thereby and the metabolites necessary for such production I can increase all my pools.
[11] Further, the PTS molecule of the present invention is a seed. Can participate in intracellular signaling pathways that can affect production yield and / or rate for one or more fine chemicals from glutamicum. For example, a protein required to introduce one or more sugars from an extracellular medium (e.g., HPr, Enzyme I, or a member of the Enzyme II complex) The process of transformation often occurs so that they can no longer enter the party. While the amount of this saccharide in the closed state of the transport system will be sufficient to maintain the normal function of the cell, the desired fine chemicals may be overproduced. Thus, the PTS protein is no longer responsive to such negative regulation so that the intracellular concentration of one or more sugars is increased, and furthermore, one or more fine chemicals are produced from the organism containing such mutant PTS protein in a more efficient or higher yield It may be desirable to modify the PTS protein of the present invention.
[12] In the present invention, for example, high energy carbon molecules such as glucose (glucose), fructose (fructose) or sucrose (sucrose) may be involved in the entry into C. glutamicum, Which provides a novel nucleic acid molecule that encodes a protein referred to herein as a phosphoenolpyruvate: sugar phosphotransferase system (PTS) protein, which may be involved in the intracellular signal transduction pathway. A nucleic acid molecule encoding a PTS protein is referred to herein as a PTS nucleic acid molecule. In a preferred embodiment, the PTS protein is a high energy carbon molecule (e. G., Glucose, fructose or sucrose) Glutamicum, and can also participate in one or more of the signaling pathways in the C. glutamicum cell. Examples of such proteins include those encoded by the genes listed in Table 1.
[13] Accordingly, one aspect of the present invention is to provide a PTS-encoding nucleic acid (e.g., DNA or RNA) as well as an isolated nucleic acid molecule (e.g., cDNA, DNA or RNA) comprising a nucleotide sequence encoding a PTS protein or a biologically active protein thereof mRNA) or a nucleic acid fragment suitable as a hybridization probe. In a particularly preferred embodiment, the isolated nucleic acid molecule is one of the nucleotide sequences described in the odd numbered sequences (eg, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: ≪ / RTI > or a complement thereof. In another particularly preferred embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence described by an odd numbered sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: , More preferably at least about 50%, preferably at least about 60%, more preferably at least about 70%, at least about 80% or at least about 90%, even more preferably at least about 95%, at least about 96%, at least about 97% 99% or more homologous nucleotide sequences. In another preferred embodiment, the isolated nucleic acid molecule encodes one of the amino acid sequences described in an even numbered sequence in the sequence listing (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8. Preferred PTS proteins of the present invention also have at least one of the PTS activities described herein.
[14] In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof, wherein the protein or portion thereof comprises an amino acid sequence that is sufficiently homologous to the amino acid sequence of the invention (e. G., An even numbered sequence in the sequence listing For example, an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention such that the protein or portion thereof retains PTS activity. Preferably, the protein encoded by the nucleic acid molecule, or a portion thereof, One or more seeds involved in influencing high energy carbon molecules (e.g., glucose, fructose or sucrose) into glutamicum. And maintains the ability to participate in the signaling pathway in glutamics cells. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60% identical to the amino acid sequence of the invention (e. G., The entire amino acid sequence selected from sequences having even number sequences in the sequence listing) , More preferably about 70%, 80% or 90%, most preferably about 95%, 96%, 97%, 98% or 99% or more homology. In another preferred embodiment, the protein comprises the entire amino acid sequence of the invention (the open reading frame shown in the corresponding odd numbered sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: Corynebacterium glutamicum protein, which is substantially homologous to the Corynebacterium glutamicum protein.
[15] In another preferred embodiment, the isolated nucleic acid molecule is selected from the group consisting of seeds. Derived from glutamicum and having about 50% or more homology with one of the amino acid sequences of the invention (e. G., One of the even numbered sequences in the sequence listing). (Or have) one or more seeds of high energy carbon molecules (eg, glucose, fructose, or sucrose) into glutamicum. A protein comprising a biologically active domain that is capable of participating in the intracellular signal transduction pathway of the glutamictic cell or having one or more of the activities set forth in Table 1 and also comprising a heterologous polypeptide or heterologous nucleic acid sequence encoding a regulatory region, For example, a PTS fusion protein).
[16] In another embodiment, the isolated nucleic acid molecule hybridizes under stringent conditions with a nucleic acid molecule that is at least 15 nucleotides in length and comprises the nucleotide sequence of the invention (e.g., an odd numbered sequence in the sequence listing). Preferably, the isolated nucleic acid molecule corresponds to a naturally occurring nucleic acid molecule. More preferably, the isolated nucleic acid is selected from the group consisting of seeds. Lt; RTI ID = 0.0 > PTS < / RTI >
[17] Another aspect of the invention relates to vectors comprising nucleic acid molecules of the invention, e. G., Recombinant vectors, and host cells into which such vectors are introduced. In one embodiment, the host cell is used to produce a PTS protein by culturing the host cell in a suitable medium. The PTS protein can then be isolated from the medium or from the host cell.
[18] Another aspect of the present invention relates to a genetic mutation microorganism into which a PTS gene has been introduced or mutated. In one embodiment, the genome of the microorganism incorporates nucleic acid molecules of the invention encoding wild-type or mutant PTS sequences as transgene and mutates. In another embodiment, the endogenous PTS gene in the genome of the microorganism is mutated, e.g., functionally, by homologous recombination with the mutated PTS gene. In another embodiment, an endogenous or introduced PTS gene in a microorganism has been mutated by one or more point mutations, deletions or inversions, but still encodes a functional PTS protein. In another embodiment, one or more regulatory regions (e. G., A promoter, repressor, or inducer) of a PTS gene in a microorganism may be engineered (e. G., Deletion, truncation, Point mutation) to regulate the expression of the PTS gene. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, particularly preferably to Corynebacterium glutamicum. In a preferred embodiment, the microorganism is also used for the production of desired compounds such as amino acids, particularly preferably lysine.
[19] In another aspect, the present invention provides a method for identifying the presence or activity of Corynebacterium diphtheria in a subject. This method involves detecting the presence or activity of Corynebacterium diphtheria in the subject, since it involves detecting one or more nucleic acid or amino acid sequences of the invention in one subject (e.g., the sequence set forth in the sequence listing as SEQ ID NOS: 1-34) can do.
[20] Another aspect of the invention relates to the isolated PTS protein or a portion thereof, for example, a biologically active site thereof. In a preferred embodiment, the isolated PTS protein, or a portion thereof, It may be involved in introducing high energy carbon molecules (such as glucose, fructose or sucrose) into glutamicum, May be involved in the intracellular signaling pathway of glutamictic cells. In another preferred embodiment, the isolated PTS protein, or a portion thereof, (Or have) one or more seeds of high energy carbon molecules (eg, glucose, fructose, or sucrose) into glutamicum. Is sufficiently homologous with the amino acid sequence of the present invention (even numbered sequence in the sequence listing) to be able to participate in the signal transduction pathway in the glutamicum cell.
[21] The present invention provides isolated preparations of PTS proteins. In a preferred embodiment, the PTS protein comprises an amino acid sequence of the invention (e. G., An even numbered sequence in the sequence listing). In another preferred embodiment, the present invention provides isolated full-length proteins substantially homologous to the entire amino acid sequence (even numbered sequence in the sequence listing) of the present invention (including an open reading frame in the corresponding odd numbered sequence in the sequence listing ). ≪ / RTI > In yet another embodiment, the protein is at least about 50%, preferably at least about 60%, more preferably at least about 70%, and most preferably at least about 80%, more preferably at least about 80% , Or 90% or more, and most preferably about 95%, about 96%, about 97%, about 98% or about 99% or more homology. In another embodiment, the isolated PTS protein comprises an amino acid sequence that is about 50% or more homologous to one of the amino acid sequences of the invention (e. G., An even numbered sequence in the sequence listing). It is capable of introducing (or having) high energy carbon molecules (such as glucose, fructose or sucrose) into the glutamicum. Can participate in the glutamictic intracellular signaling pathway, or have one or more of the activities listed in Table 1.
[22] Alternatively, the isolated PTS protein may hybridize to one of the even numbered sequences of the even numbered sequences listed in the sequence listing, or hybridize under stringent conditions, for example, at least about 50%, preferably at least about 60% Is encoded by a nucleotide sequence having about 70%, 80%, or 90%, and most preferably about 95%, about 96%, about 97%, about 98%, or about 99% And may include amino acid sequences. It is also preferred that the preferred form of the PTS protein has one or more of the PTS biological activities described herein.
[23] A PTS polypeptide or biologically active site thereof may be operably linked to a polypeptide that is not a PTS polypeptide to form a fusion protein. In a preferred embodiment, the activity of this fusion protein differs from that of the PTS protein alone. In a preferred embodiment, the fusion protein increases production yield and / or efficiency of the desired fine chemical from Corynebacterium glutamicum. In a particularly preferred embodiment, the fusion protein is introduced into a host cell to regulate the production of the desired compound from the host cell.
[24] In another aspect, the invention provides a method for screening molecules that modulate the activity of a PTS protein by interacting with the PTS protein itself, a substrate or binding partner of the PTS protein, or by modulating the transcription or translation of a PTS nucleic acid molecule of the invention .
[25] Another aspect of the present invention relates to a method for manufacturing fine chemicals. This method comprises culturing cells containing a vector expressing the PTS nucleic acid molecule of the present invention to produce a precise chemical. In a preferred embodiment, the method also comprises the step of transfecting the cells with a vector expressing a PTS nucleic acid to obtain a cell containing said vector. In another preferred embodiment, the method also includes the step of recovering fine chemicals from the culture. In a particularly preferred embodiment, the cell is selected from the genus of Corynebacterium or Brevibacterium or the strain described in Table 3.
[26] Another aspect of the invention relates to a method of regulating the production of molecules from microorganisms. Such methods include contacting the cell with a substance that modulates PTS protein activity or PTS nucleic acid expression so that the cell-associated activity is changed relative to the activity in the absence of the substance. In a preferred embodiment, the cells are regulated for the influx of one or more sugars such that the yield or rate of production of the desired fine chemicals from the microorganism is improved. The substance that modulates PTS protein activity may be a substance that stimulates PTS protein activity or PTS nucleic acid expression. Examples of substances that stimulate PTS protein activity or PTS nucleic acid expression include small molecules, active PTS proteins, and nucleic acids that encode PTS proteins introduced into cells. Examples of substances that inhibit PTS activity or expression include small molecule and antisense PTS nucleic acid molecules.
[27] Yet another aspect of the present invention relates to a method of modulating the yield of a desired compound from a cell, comprising introducing a wild-type or mutant PTS gene into the cell so as to be maintained on a separate plasmid or introduced into the genome of the host cell will be. When inserted into the genome (such insertions can be random or can occur by homologous recombination, allowing the natural gene to be replaced with the introduced copy), the production of the desired compound from the cell is regulated. In a preferred embodiment, the yield is increased. In another preferred embodiment, the chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In a particularly preferred embodiment, the amino acid is L-lysine.
[28] SUMMARY OF THE INVENTION Provides PTS nucleic acid and protein molecules that are involved in influx of high energy carbon molecules (e.g., glucose, fructose, or sucrose) into glutamicum, and that may also participate in the intracellular signaling pathway of this microorganism. The molecules of the present invention can be used to control the production of fine chemicals from microorganisms. Such regulation may be due to an increase in intracellular levels of high energy molecules required for production, such as ATP, GTP and other molecules used to cause biologic reactions that are unlikely to take place, such as the biosynthesis of fine chemicals. This regulation of fine chemicals may also be due to the fact that the degraded products of many sugars are utilized as intermediates or precursors of other biosynthetic pathways, including certain fine chemicals. Furthermore, it is known that PTS proteins are involved in specific intracellular signaling pathways that have modulatory activity on one or more of the precise chemical metabolism pathways, thereby manipulating these PTS proteins to activate the biochemical pathway of fine chemicals, The decomposition route can be suppressed. Aspects of the present invention are described in further detail below.
[29] I. Fine Chemicals
[30] The term " fine chemical substance " is well known in the art and includes molecules produced by an organism used in a variety of industries, such as, but not limited to, the pharmaceutical, agriculture and cosmetics industries. Such compounds include organic acids such as tartaric acid, itaconic acid and diaminopimelic acid, proteinaceous and nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (see, for example, Kuninaka, A. (1996) 6, Rehm et al., Eds. VCH: Weinheim), the contents of which are incorporated herein by reference), lipids, saturated and unsaturated But are not limited to, fatty acids such as arachidonic acid, diols such as propanediol and butanediol, carbohydrates such as hyaluronic acid and trehalose, aromatic compounds such as aromatic amines, vanillin and indigo, And Ong, AS, Niki, E. & Packer, L. (1996) VCH: Weinheim and references therein) 1995) " Nutrition, Lipids, Health, and Disease " Procee (published in 1995, Penang, Malaysia, AOCS Press, 1995)], enzymes, enzymes, enzymes, enzymes, (Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086) and all other chemicals listed in the references therein The metabolism and use of some of these fine chemicals will be described in more detail below.
[31] A. Amino Acid Metabolism and Uses
[32] Amino acids constitute the basic structural units of all proteins and are essential for normal cellular function in all organisms. The term " amino acid " is art recognized. Proteinogenic amino acids in amino acids are 20 species and function as structural units for proteins bound by peptide bonds, whereas nonproteinogenic amino acids (hundreds of species are known) are normally not found in proteins [Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)]. Although the L-amino acid is generally the only form found in naturally occurring proteins, the amino acid may be a D-optic or L-optic structure. The biosynthesis and degradation pathways of each of the 20 proteinaceous amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pages 578-590 ). Amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), called "essential" amino acids, are produced by the simple biosynthetic pathway, "It is easily converted to amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine). Higher animals have the ability to synthesize some of the essential amino acids, but these essential amino acids must be fed from the diet before normal protein synthesis occurs.
[33] In addition to the function of amino acids in protein biosynthesis, these amino acids are interesting chemicals themselves and many amino acids have been found to be useful in a wide variety of food, feed, chemical, cosmetic, agricultural and pharmaceutical industries. Lysine is an important amino acid not only for humans but also for the nutrition of unit animals such as poultry and pigs. Glutamate is most commonly used as a flavorant additive (mono-sodium glutamate, MSG), and aspartate, phenylalanine, glycine and citrate are widely used throughout the food industry. Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in both the pharmaceutical and cosmetic industries. Threonine, tryptophan and D / L-methionine are common feed additives (Leuchtenberger, W. (1996) Amino acids technical production and use, p. 466-502 in Rehm et al. (Eds.) Biotechnology vol. 14a, VCH: Weinheim). These amino acids also include synthetic amino acids and proteins such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan, and Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH: Weinheim, 1985, which is incorporated herein by reference in its entirety.
[34] The characteristics of biosynthesis of these natural amino acids in organisms capable of producing these natural amino acids, such as bacteria, have been well characterized (see, for example, Umbarger, HE (1978) Ann. Rev. Biochem 47: 533-606). Glutamate is synthesized by reductive amination of -Ketoglutarate, an intermediate of the citric acid cycle. Glutamine, proline and arginine, respectively, are then produced from glutamate. The biosynthesis of serine begins with 3-phosphoglycerate (the intermediate of the action) and is a three-step process that produces serine after oxidation, transamination and hydrolysis steps. Cysteine and glycine are both produced from serine, cysteine is produced by the condensation of homocysteine and serine, and glycine is produced by the transfer of side chain [beta] -carbon atoms to tetrahydrofolate in a reaction catalyzed by serine transhydroxymethylase. Phenylalanine and tyrosine are synthesized from erythrosine 4-phosphate and phosphoenol pyruvate, the precursor of the corresponding action and pentose phosphate pathway, in the other nine step biosynthetic pathway only in the final two steps after synthesis of the prephenate. Tryptophan is also synthesized from these two initial molecules, but its synthesis is an eleven step pathway. Tyrosine may also be synthesized from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine and leucine are all biosynthetic products of pyruvate, the end product of the action. Aspartate is formed from oxaloacetate, which is an intermediate of the citric acid cycle. Asparagine, methionine, threonine and lysine, respectively, are produced by the conversion of aspartate. Isoleucine is formed from threonine. In the complex 9-step pathway, histidine is generated from the activated 5-phospholibosyl-1-pyrophosphate.
[35] Amino acids exceeding the protein synthesis requirement of the cell can not be stored and instead are degraded and provided as intermediates for the major metabolic pathway of the cell (see, for example, Stryer, L. Biochemistry 3rd ed. Degradation and the Urea Cycle ", p. 495-516 (1988)). Even though cells can convert unnecessary amino acids into useful metabolic intermediates, amino acid production is a lossy synthesis considering energy, precursor molecules, and the enzymes required to synthesize these amino acids. Thus, it is not surprising that the presence of a particular amino acid modulates the biosynthesis of amino acids by feedback inhibition, which slows or completely aborts its own production. (The overall content of the feedback mechanism of the amino acid biosynthetic pathway is described in Stryer, L. et al. Biochemistry 3rd ed. Ch. 24 " Biosynthesis of Amino Acids and Heme " p. 575-600 (1988)). Thus, the yield of any particular amino acid is limited by the amount of amino acid present in the cell.
[36] B. Metabolism and uses of vitamins, cofactors and nutrients
[37] Vitamins, cofactors, and nutrients constitute another group of molecules that higher animals need to ingest, losing their ability to synthesize, even if they are easily synthesized by other organisms such as bacteria. These molecules are either living materials themselves or precursors of biologically active substances that can act as electron carriers or intermediates in a variety of metabolic pathways. In addition to their nutritional value, these compounds have considerable industrial value as pigments, antioxidants and catalysts, or as adjuvants in other processes (for their structure, activity and industrial applications see, for example, Ullman's Encyclopedia of Industrial The term " vitamin " is recognized in the art and is necessary for the normal functioning of an organism, but the organism is itself synthesized (see, for example, " Vitamins ", vol. A27, p. 443-613, VCH: Weinheim, The term " cofactor " includes non-proteinogenic compounds necessary for normal enzyme activity to occur. Such compounds may be organic or inorganic in nature It is preferred that the cofactor molecule of the invention is organic. The term " nutrient " refers to a plant or animal, Examples of such molecules include vitamins, antioxidants, and some lipids (e.g., polyunsaturated fatty acids).
[38] The characteristics of biosynthesis of these molecules in organisms capable of producing them, such as in bacteria, have been well characterized (Ullman ' s Encyclopedia of Industrial Chemistry, " Vitamins " vol. A27, p. 443-613, VCH: Weinheim, Proceedings of "Nutrition, Lipids, Health, and Disease" (1995) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Ong, AS, Niki, E. & Packer, L. 1994, at Peninsular Malaysia, AOCS Press, Champaign, IL X, 374 S). In this study, we have investigated the effect of differentiation on the microbial pathogenesis.
[39] Thiamine (vitamin B ₁ ) is produced by chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin B ₂ ) is synthesized from guanosine-5'-triphosphate (GTP) and ribose-5'-phosphate. Riboflavin is then used in the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). A group of compounds, collectively termed " vitamin B ₆ " (e.g. pyridoxine, pyridoxamine, pyridoxine-5'-phosphate and commercially available pyridoxine hydrochloride) Methylpyridine. Pantothenate (pantothenic acid, (R) - (+) - N- (2,4-dihydroxy-3,3-dimethyl-1-oxobutyl) - - alanine) is produced by chemical synthesis or fermentation . The final step in the biosynthesis of pantothenate consists of ATP-induced condensation of β-alanine and pantoic acid. Enzymes involved in the biosynthesis step to convert to pantothenic acid, the biosynthetic step to convert to -Alanine, and the condensation to pantothenic acid are known. The metabolic activation form of pantothenate is coenzyme A. The biosynthesis for this coenzyme A proceeds by a 5-step enzyme reaction. Pantothenate, pyridoxal-5'-phosphate, cysteine, and ATP are precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate but also (R) -pantotic acid, (R) -phantolactone, (R) -Panthenol (provitamin B ₅ ), pantetaine A is also catalyzed.
[40] The biosynthesis of biotin from pimelyl-CoA, a precursor molecule in microorganisms, has been studied in detail and various related genes have been identified. A number of corresponding proteins have also been found to be involved in Fe-cluster synthesis, and these proteins are members of the nifS class proteins. Lipoic acid is derived from octanoic acid and acts as a coenzyme in energy metabolism, which is part of the pyruvate dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex in energy metabolism. Folates are all a group of derivatives of folic acid and folic acid is derived from L-glutamic acid, p-amino-benzoic acid and 6-methylpterin. Biosynthesis of folic acid and its derivatives, starting from the metabolic intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and p-amino-benzoic acid has been studied in detail in some microorganisms.
[41] Corinoids (e.g., cobalamin and especially vitamin B ₁₂ ) and porphyrin belong to the family of chemicals characterized by the tetrapyrrole ring system. The biosynthesis of vitamin B ₁₂ is complex enough in that many enzymes and substrates are currently known, although their characteristics are not fully characterized. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives also called "niacin". Niacin is an important coenzyme, NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate) and their reduced form precursors.
[42] Mass production of these compounds is largely dependent on cell-free chemistry, although some of these chemicals, such as riboflavin, vitamin B ₆ , pantothenate and biotin, are also produced by large-scale cultivation of microorganisms. Only vitamin B ₁₂ is produced entirely by fermentation, which is due to the complexity of the synthesis of vitamin B ₁₂ . In vitro methodology requires considerable input of materials and time, often a lot of cost.
[43] C. Metabolism and Uses of Purines, Pyrimidines, Nucleosides and Nucleotides
[44] Purine and pyrimidine metabolite genes and their corresponding proteins are important targets in the treatment of tumor diseases and viral infections. The term " purine " or " pyrimidine " includes a nitrogen-containing base which is a component of nucleic acids, coenzymes and nucleotides. The term " nucleotide " includes basic structural units of a nucleic acid molecule consisting of a nitrogen-containing base, a pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA, the sugar is D-deoxyribose) and phosphoric acid. The term " nucleoside " includes molecules which act as precursors of the nucleotide but which do not have the phosphoric acid residue of the nucleotide. By inhibiting the biosynthesis of these molecules or by inhibiting the mobilization of these molecules to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; By inhibiting the activity by targeting to cancer cells, the ability to divide and replicate tumor cells can be suppressed. There are also nucleotides that act as energy stores (i.e., AMPs) or coenzymes (i.e., FAD and NAD) rather than forming nucleic acid molecules.
[45] It has been described in various publications that these chemicals can be used for the above medical symptoms by affecting purine and / or pyrimidine metabolism (see, for example, Christopherson, RI and Lyons, SD (1990) "Potent inhibitors of de Novo pyrimidine and purine biosynthesis as chemotherapeutic agents. " Med. Res. Reviews 10: 505-548). Studies of enzymes involved in purine and pyrimidine metabolism have focused on the development of new drugs that can be used, for example, as immunosuppressive or antiproliferative agents (Smith, JL, (1995) Enzymes in nucleotide synthesis. Curr. Opin. Struct Biol., 5: 752-757; (1995) Biochem Soc. Transact., 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides can be used as intermediates in the biosynthesis of various fine chemicals (e.g., thiamine, S-adenosyl-methionine, folate or riboflavin) For use as a carrier (for example, ATP or GTP), and for chemicals that are commonly used as perfuming agents (e.g., IMP or GMP) or for various medical uses (See, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology, 6, Rehm et al., Eds VCH: Weinheim, p. In addition, there is an increasing research on the development of crop protection chemicals, including fungicides, herbicides and insecticides, using enzymes involved in purine, pyrimidine, nucleoside or nucleotide metabolism as targets.
[46] The characterization of the metabolism of these compounds in bacteria has been identified (see, for example, Zalkin, H. and Dixon, JE (1992) "De novo purinenucleotide biosynthesis", in Progress in Nucleic Acid Research and Molecular Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York), " Nucleotides and Nucleosides ", Chapter 8 in Biochemical Pathways, Vol. 42, Academic Press, ). Purine metabolism is the subject of intensive research and is essential for the normal functioning of cells. Damaged purin metabolism in higher animals can lead to serious illnesses such as gout. The purine nucleotides are synthesized in a series of steps via inosine-5'-phosphate (IMP), an intermediate compound that produces guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate Phosphates, and triphosphates used as nucleotides from these GMPs or AMPs are easily formed. Because these compounds are also used as energy stores, their degradation provides energy for a number of diverse biosynthetic processes in the cell. Pyrimidine biosynthesis is driven by the formation of free-5'-monophosphate (UMP) from ribose-5-phosphate. The UMP is then converted to cytidine-5'-triphosphate (CTP). The deoxy form of all these nucleotides is generated in a one-step reduction reaction in which the diphosphate ribose form of the nucleotide is converted to the diphosphate deoxyribose form of the nucleotide. During phosphorylation, these molecules can participate in DNA synthesis.
[47] D. Metabolism and Uses of Trehalose
[48] Trehalose is composed of two sugar molecules bound by α, α-1,1 linkages. Trehalose is commonly used in the food and beverage industries as an additive for sweeteners, dried or frozen foods. However, it is also used in the pharmaceutical, cosmetic and biotechnology industries (see, for example, Nishimoto et al., (1998) US Pat. No. 5,759,610; Singer, MA and Lindquist, S. (1998) Trends Biotech. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-10). Trehalose is produced by enzymes from many microorganisms and released naturally into the surrounding medium, which can be obtained from the medium using methods known in the art.
[49] II. Phosphoenolpyruvate: a sugar phosphotransferase system
[50] The ability of a cell to grow and rapidly divide during culture is largely determined by the amount that the cell can enter and use high energy molecules such as glucose and other saccharides. Different transport proteins exist to transport different carbon sources within the cell. These include sugar transport proteins such as glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose or raffinose and transport proteins of starch or cellulose degradation products. Other transport systems include those for alcohols (e.g., methanol or ethanol), alkanes, fatty acids, and organic acids (e.g., acetic acid or lactic acid). In bacteria, sugars can be transported into cells across the membrane through a variety of mechanisms. In addition to the symport of the sugar that moves with the hydrogen ion, the most commonly used process in bacteria for sugar infusion is the phosphoenolpyruvate: sugar phosphotransferase system (PTS). The system not only catalyzes the transfer of sugar and hexitol (which occurs simultaneously with phosphorylation), but also regulates cell metabolism when carbohydrates are available. These PTS systems are ubiquitous in bacteria, but not in aqua bacteria or eukaryotic cells.
[51] Functionally, the PTS system consists of two cytoplasmic proteins, Enzyme I and HPr, and a number of sugar-specific intrinsic and extrinsic membrane transport complexes, each of which has a unique sugar on it, II) ', for example,' Enzyme II ^Glu 'is an enzyme complex that binds to glucose. Enzyme II specific to monosaccharides, disaccharides or polysaccharides such as glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose and raffinose is known. Enzyme I transfers phosphate groups from phosphoenolpyruvate (PEP) to HPr, the phosphate transport protein. HPr then transfers the phosphate group to another Enzyme II transport complex. The amino acid sequences of Enzyme I and HPr are very similar in all bacteria, but the PTS transporter sequences can be classified as those that are not structurally related to them. Furthermore, the number and homology of these genes vary from one bacteria to another. this. The E. coli genome encodes 38 different PTS proteins, 33 of which are subunits belonging to 22 different transporters. M. The M. genitalium genome contains one gene for each of Enzyme I and HPr, and contains only two PTS transporter genes. tea. Palladium (T. palladium) and seeds. The genome of C. trachomatis contains the genes for the Enzyme I and HPr-like proteins, but no PTS transporter.
[52] All PTS transporters consist of three functional units, IIA, IIB and IIC, which appear as domains of protein subunits (eg IIA ^Glc IICB ^Glc ) or domains of a single polypeptide chain (IICBA ^GlcNAc ) in the complex. IIA and IIB, in turn, transfer phosphate groups from HPr to the sugar moiety. The IIC contains sugar binding sites and crosses the lining six or eight times. The transfer of sugar takes place with provisional phosphorylation of the IIB domain. Enzyme I, HPr, and IIA are phosphorylated at histidine residues, while IIB subunits are phosphorylated at cysteine or histidine residues, depending on the particular transporter involved. Phosphorylation of the incoming sugars has the advantage of preventing the sugars from diffusing back into the extracellular medium through the cell membrane, because the charged phosphate groups can not easily traverse the hydrophobic center of the membrane.
[53] Some PTS proteins are not only active in the active transport of sugars, but also play a role in intracellular signal transduction. These subunits regulate their targets by other steric hindrance or phosphorylation. Its regulatory activity varies according to its degree of phosphorylation (i. E., The ratio of non-phosphorylated vs. phosphatized), and the rate of sugar-dependent dephosphorylation and phosphoenolpyruvate-dependent reoxidation. this. Examples of such intracellular regulation by PTS proteins in E. coli include the inhibition of glycerol kinase by dephosphorylated IIA ^Glu and the activation of adenylate cyclase by the phosphorylated form of this protein. Also, within these microorganisms, the HPr and IIB domains of some transporters regulate gene expression by reversibly phosphorylating transcription termination factors. In Gram-positive bacteria, the activity of HPr is regulated by HPr-specific serine kinases and phosphatases. For example, HPr phosphorylated at serine 46 functions as an inhibitory complement of transcriptional repressor CcpA. Finally, the non-phosphorylated enzyme I inhibits the sensor kinase, CheA, in the bacterial chemotaxis, allowing direct association of glycosyltransferases with systems that control the transport of bacteria and the transport of bacteria (Sonenshein, AL, et al, eds Bacillus subtilis and other gram-positive bacteria ASM:... Washington, DC], reference [Neidhardt, FC, et al, eds (1996) Escherichia coli and Samonella ASM Press:... Washington, DC], See, for example, Lengeler et al ., (1999) Biology of Prokaryotes., Section II, pp. 68-87, Thieme Verlag: Stuttgart).
[54] III. Elements and Methods of the Invention
[55] The present invention is based, in part, on the ability of high energy carbon molecules (e. G., Glucose, fructose or sucrose) to be involved in the entry into C. glutamicum and to participate in one or more intracellular signaling pathways of this microorganism Based on the discovery of new molecules and protein molecules referred to herein as PTS nucleic acids. In one embodiment, the PTS molecule functions to infuse high-energy carbon molecules into the cell and obtains energy that can be used to cause biochemical reactions that are unlikely to occur energetically due to degradation of the high-energy carbon molecules in the cell , The degradation products can act as intermediates and precursors for a number of metabolic pathways. In another embodiment, the PTS molecule participates in one or more cell signaling pathways, and a modified form of the PTS molecule (e.g., a phosphorylated PTS protein) may be involved in signal transduction cascades that modulate one or more cellular activities. In a preferred embodiment, the activity of the PTS molecule of the present invention affects the production of the desired fine chemical by this organism. In a particularly preferred embodiment, the PTS molecule of the present invention is selected from the group consisting of seeds. Its activity is regulated so that production yield, yield and efficiency for one or more fine chemicals from glutamicum are regulated.
[56] The term " PTS protein " or " PTS polypeptide " refers to a high energy carbon compound (such as monosaccharides, disaccharides or polysaccharides such as fructose, mannose, sucrose, glucose, raffinose, galactose, ribose, lactose, And includes proteins involved in influx into cells from the extracellular medium. Such proteins may also participate in one or more intracellular signaling pathways, such as, but not limited to, directing intracellular entry of other sugars. Examples of PTS proteins include the PTS gene described in Table 1 and those encoded by the sequence described as odd-numbered in the sequence listing. A general reference to a PTS system is provided in Stryer, L. (1988) Biochemistry. Chapter 37: "Membrane Transport", W.H. Freeman: New York, p. 959-961], Darnell, J. et al. (1990) Molecular Cell Biology Scientific American Books: New York, p. 525-553 and Michal, G., ed. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Chapter 15 "Special Bacterial Metabolism". The term " PTS gene " or " PTS nucleic acid sequence " includes a nucleic acid sequence encoding a PTS protein, which consists of a coding region and its corresponding non-translational 5 'and 3' sequence regions. Examples of PTS genes include those listed in Table 1. The term " yield " or " productivity " is art-recognized and refers to the concentration of a culture product (e.g., the desired fine chemical) formed within a given volume of culture, . The term " production efficiency " includes the time required to reach a certain target production level. The term " yield " or " product / carbon yield " is well known in the art and includes the efficiency with which a carbon source is converted into a product (i.e., a fine chemical). This is generally expressed as the product of, for example, kg per unit of carbon source per kg. As the yield or yield of the compound increases, the yield of molecules of the compound or the yield of useful molecules increases over a given period of time in the amount of a given culture medium. The term " biosynthesis " or " biosynthetic pathway " is a recognized term in the art and includes the synthesis of a compound, preferably an organic compound, from an intermediate compound by a cell and can be a multistage, highly regulated process. The term " degradation " or " degradation pathway " is a recognized term in the art and includes the degradation of a compound, preferably an organic compound, by a cell, generally a molecule of smaller or less complex structure , It can be a multistage, highly regulated process. Metabolism of certain compounds (e.g., metabolism of amino acids such as glycine) then includes the entire biosynthesis, modification, and degradation pathways of the cells involved in these compounds. The term " transport " or " inflow " includes promoting movement through cell membranes known in the art and which one or more molecules can not otherwise pass through.
[57] In another embodiment, the PTS molecule of the present invention is a PTS molecule. In microorganisms such as glutamicum, production of desired molecules such as fine chemicals can be controlled. Using recombinant techniques, one or more of the PTS proteins of the invention can be engineered to regulate function. For example, a protein involved in the PTS-mediated glucose entry may be modified to optimize its activity, and thus the PTS system for glucose uptake can transfer increased amounts of glucose into the cell. Glucose molecules are used not only to produce biochemical reactions that are not as energetically as the biosynthesis of fine chemicals, but also as precursors and intermediates in the biosynthetic pathway of many fine chemicals (eg, serine from 3-phosphoglycerate Lt; / RTI > In each case, the overall yield or production rate of one of the desired fine chemicals may be increased by increasing the amount of energy available for such production, or by increasing the availability of the compound necessary for such production to occur.
[58] Furthermore, many PTS proteins are known to play an important role in intracellular signaling pathways that regulate cellular metabolism and glucose uptake to maintain the availability of carbon sources. For example, an increase in the intracellular level of fructose 1,6-bisphosphate (a compound produced during the action) leads to phosphorylation at the serine residue of HPr, which acts as a phosphorylated donor in any PTS transport Thereby preventing further sugar inflow. By structurally activating HPr by mutating such that the serine residue of HPr is not phosphorylated, thereby increasing the intracellular sugar flux, thereby increasing the intracellular energy storage and intermediates / precursors for the biosynthesis of one or more desired fine chemicals .
[59] The isolated nucleic acid sequence of the present invention is contained within the genome of the Corynebacterium glutamicum strain, which can be sold in the American Type Culture Collection under the name ATCC13032. Isolated seed. Nucleotide sequence and seed of glutamicum PTS DNA. The predicted amino acid sequences of the glutamicum PTS protein are shown in odd and even sequence numbers, respectively, in the sequence listing.
[60] This nucleotide sequence was subjected to computer analysis to identify and / or identify it as a sequence encoding a metabolic pathway protein.
[61] The present invention also relates to a protein having an amino acid sequence that is substantially homologous to the amino acid sequence of the present invention (e.g., the sequence of the even sequence number in the sequence listing). As used herein, a protein having an amino acid sequence that is substantially homologous to the amino acid sequence selected is about 50% or more homologous to the selected amino acid, e. G., The entire amino acid sequence selected. A protein having an amino acid sequence that is substantially homologous to the amino acid sequence selected is about 50 to 60% or more, preferably about 60 to 70% or more, more preferably about 70 to 80%, more preferably about 80 to 80% 90%, or 90% to 95% or more, and most preferably about 96% or more, 97% or more, 98% or more, 99% or more.
[62] The PTS protein of the present invention or a biologically active portion or fragment thereof may be produced by a variety of methods. It may carry a high energy carbon-containing molecule in glutamicum, or be involved in intracellular signal transduction in the organism, or have one or more of the activities listed in Table 1. [
[63] Various aspects of the invention are described in more detail in the subsections below.
[64] A. Isolated nucleic acid molecules
[65] One aspect of the present invention is the use of the isolated nucleic acid molecule encoding a PTS polypeptide or a biologically active portion thereof as a hybridization probe or primer for identification or amplification of a PTS-encoding nucleic acid (e. G., PTS DNA) Lt; / RTI > nucleic acid fragments. As used herein, the term " nucleic acid molecule " includes DNA or RNA analogs that are generated using DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), and nucleotide analogs It means. The term also includes untranslated sequences located at both the 3 ' and 5 ' ends of the coding region of the gene, about 100 nucleotide sequences upstream from the 5 ' end of the coding region, And about 20 or more nucleotide sequences downstream from the terminus. The nucleic acid molecule may be single stranded or double stranded, preferably double stranded DNA. An " isolated " nucleic acid molecule is one that is separate from other nucleic acid molecules present in the natural source of the nucleic acid. Preferably, the " isolated " nucleic acid has no sequence on both sides of the native nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., the sequence located at the 5 'and 3' ends of the nucleic acid). For example, in various embodiments, the isolated PTS nucleic acid molecule is present in the genomic DNA of the cell from which it is derived (e. G., Glutamicum cells), about 5 kb, 4 kb , 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence. In addition, " isolated " nucleic acid molecules, e. G., DNA molecules, when produced by recombinant techniques do not actually contain other cellular materials or culture media, or chemical precursors or other chemicals when chemically synthesized .
[66] Using standard molecular biology techniques and the sequence information provided herein, nucleic acid molecules of the invention, e. G., Nucleic acid molecules having a nucleotide sequence of odd numbered SEQ ID NOs in the sequence listing, or a portion thereof, may be isolated. For example, all or a portion of one of the sequences of the odd sequence numbers in the sequence listing can be used as a hybridization probe and hybridized using standard hybridization techniques (e.g., Sambrook, J., Fritsh, EF, and Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Mr. glutamicum library. Glutamicum PTS DNA can be isolated. Further, the nucleic acid sequence of the present invention (for example, the sequence of the odd-numbered sequence of the present invention (for example, the sequence of the odd-numbered sequence of the present invention) by a polymerase chain reaction using oligonucleotide primers designed on the basis of this same sequence ) Can be isolated from a nucleic acid sequence of the present invention (for example, a sequence of an odd-numbered sequence number (for example, an oligonucleotide sequence of an odd-numbered sequence number ). ≪ / RTI > For example, mRNA can be isolated from normal endothelial cells (e.g., by the guanidinium-thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) Can be obtained from reverse transcriptase (e. G., Moloney MLV reverse transcriptase available from Gibco / BRL (Bethesda, MD) or from Seikagaku America, Inc., St. Petersburg, FL) Lt; RTI ID = 0.0 > AMV < / RTI > reverse transcriptase). A synthetic oligonucleotide primer for polymerase chain reaction amplification can be designed based on one of the nucleotide sequences listed in the Sequence Listing. The nucleic acid of the present invention can be amplified using cDNA or, alternatively, genomic DNA as a template and using appropriate oligonucleotide primers according to standard PCR amplification techniques. The amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequencing. Oligonucleotides corresponding to the PTS nucleotide sequence can also be prepared using standard synthetic techniques, for example, an automated DNA synthesizer.
[67] In a preferred embodiment, the isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences listed in the Sequence Listing. The nucleic acid sequence of the present invention as described in the Sequence Listing corresponds to the Corynebacterium glutamicum PTS DNA of the present invention. This DNA contains a sequence encoding a PTS protein (i.e., a "coding region" shown in the sequence of odd-numbered sequences in the sequence listing) as well as a 5'-untranslated sequence and a 3'-untranslated sequence shown in the sequence of each odd- Sequence. Alternatively, the nucleic acid molecule may comprise only one coding region of the nucleic acid sequence of the sequence listing.
[68] RXA, RXN, RXS or RXC for identification with five numerals followed by the designations "RXA", "RXN", "RXN", "RXS" or "RXC" (I. E., RXA01503, RXN01299, RXS00315 or RXC00953). Each of the nucleic acid sequences consists of three parts, the 5 'upstream region, the coding region and the downstream region. To avoid confusion, each of these three regions is identified by the same RXA, RXN, RXS or RXC designation. In addition, " one of the odd sequences of the sequence listing " means any of the nucleic acid sequences in the sequence listing, and may be distinguished by their other RXA, RXN, RXS or RXC names. The coding region of each of these sequences is also translated into the corresponding amino acid (the corresponding nucleic acid sequence is described immediately below), which is even numbered in the sequence listing. For example, the coding region for RXA02229 is set forth in SEQ ID NO: 1, and the amino acid sequence it codes for is set forth in SEQ ID NO: 2. The sequences of the nucleic acid molecules of the present invention are identified by the same RXA, RXN, RXS or RXC designation as the amino acid molecules they encode so that they can be easily correlated. For example, the amino acid sequences designated RXA01503, RXN01299, RXS00315, and RXC00953 are translations of the coding regions of the nucleotide sequences of the nucleic acid molecules RXA01503, RXN01299, RXS00315 and RXC00953, respectively. The correspondence between the nucleotide and amino acid sequences of RXA, RXN, RXS and RXC of the present invention and their assigned SEQ ID NO. For example, as shown in Table 1, the nucleotide sequence of RXN01299 is shown in SEQ ID NO: 7, and the amino acid sequence of RXN01299 is shown in SEQ ID NO: 8.
[69] Some of the genes of the present invention are " F-nomenclature genes ". F-name genes include the genes listed in Table 1, which have an "F" in front of the RXA, RXN, RXS, or RXC designations. For example, as shown in Table 1, SEQ ID NO: 3 named "F RXA00315" is an F-designation gene and SEQ ID NO: 9, 11 and 13 ("F RXA01299", "F RXA01883" Quot; RXA01889 ").
[70] In one embodiment, the nucleic acid molecule of the present invention comprises the seeds described in Table 2. It does not include the nucleic acids of glutamicum. For the dapD gene, the sequence for this gene can be found in Wehrmann, A., et al , (1998) J. Bacteriol . 180 (12): 3159-3165. However, the sequences obtained by the present inventors are considerably longer than the published sequence form. It is believed that the published sequence form is dependent on the incomplete initiation codon and therefore only represents a fragment of the actual coding region.
[71] In another preferred embodiment, the isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of the invention (e. G., An odd sequence numbered sequence in the sequence listing). The complementary nucleic acid molecule for one of the nucleotide sequences shown in the present invention is sufficiently complementary to one of the nucleotide sequences shown in the sequence listing (for example, the sequence of the odd-numbered sequence numbers), so that one of the nucleotide sequences of the present invention By hybridization, a stable double strand is formed.
[72] In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises about 50%, 51%, 52%, 53%, or about 50% of the nucleotide sequence of the invention (e.g., one of the sequences of the odd sequence numbers in the sequence listing) , 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably about 61%, 62%, 63%, 64%, 65%, 66%, 67% , 69%, 70%, or more preferably about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94% , 97%, 98%, or 99% or more homologous to the nucleotide sequence of SEQ ID NO. Moderate ranges and identity values (e.g., 70-90% identical or 80-95% identical) to the above-mentioned ranges are included in the present invention. For example, an identity value range using any combination of the values referred to in the above as the upper limit and / or lower limit is included in the present invention. In a further preferred embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence that hybridizes under stringent conditions, for example, to one or a portion of the nucleotide sequence of the invention (e.g., the sequence of the odd sequence numbering of the sequence listing) .
[73] In addition, the nucleic acid molecule of the present invention may comprise only a portion of the coding region of one of the sequences of the odd-numbered sequence of the Sequence Listing, for example, a fragment that can be used as a probe or primer, or a fragment encoding a biologically active portion of the PTS protein Fragments. ≪ / RTI > Seed. Use of the nucleotide sequence determined from the cloning of the PTS gene from glutamicum to identify and / or clone PTS homologues from other Corynebacteria or related species as well as PTS homologs in other types of cells and organisms Probes and primers designed for use. Probes / primers typically comprise substantially purified oligonucleotides. Oligonucleotides are typically linked under stringent conditions to a sense strand of one of the nucleotide sequences of the invention (e.g., one of the sequences of the odd sequence numbers of the sequence listing), an anti-sense sequence of one of these sequences, And includes a nucleotide sequence region that hybridizes with about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of its variants. A primer based on the nucleotide sequence of the present invention can be used in a PCR reaction for cloning a PTS homologue. Probes based on PTS nucleotide sequences can be used to find transcripts or genomic sequences encoding the same or homologous proteins. In a preferred embodiment, the probe further comprises a labeling group attached thereto, for example the labeling group may be a radioactive isotope, a fluorescent compound, an enzyme or an enzyme auxiliary. Such probes can be used to detect cells that mis-express the PTS protein, for example, by measuring the level of PTS-encoding nucleic acid in a cell sample, for example by detecting the level of PTS mRNA or by determining whether the genomic PTS gene has been mutated or deleted And can be used as part of a diagnostic test kit for identification.
[74] In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof comprising an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (e. G., The sequence of the even sequence number in the sequence listing) Protein or part thereof is seed. Maintains the ability to participate in the transport of high energy carbon molecules (e.g., glucose) into glutamicum, and can also participate in one or more intracellular signaling pathways. As used herein, the term " fully homologous " refers to a minimal number of amino acid residues of the same or equivalent to the amino acid sequence of the invention (e.g., one amino acid residue in one of the even numbered sequences of the sequence listing Amino acid residues having a side chain similar to the amino acid sequence of SEQ ID NO: 2), or a portion thereof. It is capable of transporting high energy carbon-containing molecules such as glucose into glutamicum, and can also participate in intracellular signal transduction in this microorganism. Protein members of this metabolic pathway, as described herein, It is capable of transporting high energy carbon-containing molecules such as glucose into glutamicum, and can also participate in intracellular signal transduction in this microorganism. Examples of such activities are also described herein. Thus, the term " function of the PTS protein " refers to the ability of one or more phosphoenolpyruvate-based sugar transport pathways to contribute to the overall function and / or control of the pathway and / ) Contributes directly or indirectly to production efficiency. Examples of PTS protein activity are listed in Table 1.
[75] In yet another embodiment, the protein is at least about 50% to about 60%, preferably at least about 60% to about 70%, more preferably at least about 60%, more preferably at least about 60% Homology is about 70% to 80%, 80% to 90%, 90% to 95%, and most preferably about 96%, 97%, 98%, or 99% homology.
[76] A portion of the protein encoded by the PTS nucleic acid molecule of the invention is preferably a biologically active portion of one of the PTS proteins. As used herein, the term " a biologically active portion of a PTS protein " Can carry a high energy carbon-containing molecule such as glucose in glutamicum, or can be involved in intracellular signaling of this microorganism, or can be part of a PTS protein having activity as described in Table 1, , And its domain / motif. PTS protein or a portion thereof which is biologically active. An assay of enzyme activity can be performed to determine whether it is capable of participating in the transport of high energy carbon-containing molecules such as glucose in glutamicum, or can be involved in intracellular signal transduction of this microorganism. Such analytical methods are well known to those skilled in the art, as described in Example 8.
[77] Other nucleic acid fragments encoding a biologically active portion of the PTS protein may be derived from the amino acid sequence of the invention (e. G., By recombinant expression in vitro), such as by expressing the coding portion of a PTS protein or peptide Lt; / RTI > sequence), and assessing the activity of the coding portion of the PTS protein or peptide.
[78] The present invention relates to a method for producing a PTS protein which is different from one of the nucleotide sequences shown in the present invention (for example, the sequence of odd-numbered SEQ ID NOs in the Sequence Listing), but which is encoded by the nucleotide sequence of the present invention due to degeneracy of the genetic code (And fragments thereof). In another embodiment, the isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having the amino acid sequence shown in the sequence listing (e. G., Sequence of even sequence number). In another embodiment, the nucleic acid molecule of the present invention is a full-length seed which is substantially homologous to the amino acid sequence of the present invention (encoded by the open reading frame shown in the sequence of the odd sequence numbers in the Sequence Listing). Lt; RTI ID = 0.0 > glutamic < / RTI >
[79] It will be understood by those skilled in the art that in one embodiment the sequences of the present invention do not include sequences of the prior art, such as the Genbank sequences described in Tables 2 or 4, which were previously available for the present invention. In one embodiment, the invention contemplates the use of a nucleic acid sequence that is identical to a nucleotide or amino acid sequence of the present invention that is larger than the sequence of the prior art (e. G., The gene sequence described in Tables 2 or 4 (or a protein encoded by such sequence) %) ≪ / RTI > and an amino acid sequence. For example, the present invention encompasses nucleotide sequences that are larger (greater than) or greater than 44% identical to the nucleotide sequence designated RXA01503 (SEQ ID NO: 5), greater than the nucleotide sequence designated RXA00951 Or greater) and a nucleotide sequence that is greater than (greater than or equal to) 38% of the nucleotide sequence designated RXA01300 (SEQ ID NO: 21) and a nucleotide sequence that is at least 41% identical thereto. Those skilled in the art will be able to examine the GAP-calculated percent identity score shown in Table 4 for each of the three highest hits for a given sequence and calculate the GAP-calculated highest identity (% You can calculate the lower bound threshold of identity (%) for a given sequence. Those skilled in the art will also appreciate that greater percent identity (e.g., about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% Or 60% or more, preferably about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% or more, more preferably about 71% %, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 89%, or 90%, or 91%, 92%, 93%, 94%, or even more preferably about 95%, 96%, 97%, 98%, or 99% And amino acid sequences are included in the present invention.
[80] Those skilled in the art will appreciate that seeds described by odd numbered SEQ ID NO. In addition to the glutamicum PTS nucleotide sequence, it will be appreciated that the polymorphism of the DNA sequence resulting in a change in the amino acid sequence of the PTS protein may be present in a population (e. G., The glutamicum population). This genetic polymorphism in the PTS gene can exist between individuals within a population due to natural changes. As used herein, the terms " gene " and " recombinant gene " refer to a PTS protein, Refers to a nucleic acid molecule comprising an open reading frame encoding a glutamicum PTS protein. These natural changes typically change the nucleotide sequence of the PTS gene by 1 to 5%. Any and all such nucleotide changes in the PTS gene, which result from a natural change and do not alter the functional activity of the PTS protein, and the resulting polymorphism of the amino acid are within the scope of the present invention.
[81] Natural variants and seeds of the present invention. Non-seed of glutamicum PTS DNA. The nucleic acid molecule corresponding to the glutamicum homologue is the one disclosed in the present application. Based on their homology to the glutamicum PTS nucleic acid, the hybridization probes can be used as hybridization probes according to standard hybridization techniques under stringent hybridization conditions. Can be isolated using glutamicum DNA or a portion thereof. Thus, in another embodiment, the isolated nucleic acid molecule of the present invention hybridizes with a nucleic acid molecule comprising a nucleotide sequence of odd numbered SEQ ID NOs in the Sequence Listing under stringent conditions, with a length of at least 15 nucleotides. In another embodiment, the nucleic acid is at least 30, 50, 100, or 250 nucleotides in length. As used herein, the term " hybridizing under stringent conditions " is meant to describe conditions for hybridization and washing, wherein at least 60% homologous nucleotide sequences relative to each other remain hybridized to each other. Preferably, the conditions are those in which sequences that are usually about 65% or more, more preferably about 70% or more, and even more preferably about 75% or more homologous to each other remain hybridized to each other. Such stringent conditions are well known to those skilled in the art and can be found in Ausubel et al ., Current Protocols in Molecular Biology , John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A preferred non-limiting example for stringent hybridization conditions is one or more washes in 0.2 x SSC, 0.1% SDS at 50-65 < 0 > C after hybridization in 6 x sodium chloride / sodium citrate (SSC) at about 45 < 0 > C. Preferably, the isolated nucleic acid molecule of the invention that hybridizes under stringent conditions with the nucleotide sequence of the invention corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule that has a nucleotide sequence that is naturally occurring (eg, encodes a natural protein). In one embodiment, the nucleic acid is a natural seed. It encodes the glutamicum PTS protein.
[82] In addition to the naturally occurring variants of the PTS sequence that may be present in a population, those skilled in the art will also appreciate that variations in the nucleotide sequence of the present invention are not altered by altering the amino acid sequence of the encoded PTS protein without altering the functionality of the PTS protein Can be introduced. For example, nucleotide substitutions that result in amino acid substitutions in " non-essential " amino acid residues can occur in the nucleotide sequences of the present invention. Quot; non-essential amino acid " residue is a residue that can be changed from the wild-type sequence of one of the PTS proteins (e. G., Sequences of even sequence numbers in the sequence listing) without altering the activity of the PTS protein, Residues are required for PTS protein activity. However, other amino acid residues (e. G., Residues that are not conserved or are conserved in a domain having PTS activity) may not be required for activity and thus may be altered without altering PTS activity.
[83] Thus, another aspect of the invention relates to nucleic acid molecules that encode a PTS protein that includes changes in amino acid residues that are not essential for PTS activity. Such a PTS protein differs in amino acid sequence from the sequence of the even-numbered sequence in the sequence listing and has at least one of the PTS activities described herein. In one embodiment, the isolated nucleic acid molecule comprises an amino acid sequence that is at least about 50% homologous to the amino acid sequence of the invention. A nucleotide sequence encoding a protein capable of transporting a high energy carbon-containing molecule such as glucose into glutamicum, or capable of participating in intracellular signal transduction of the microorganism or having at least one activity as described in Table 1 . Preferably, the protein encoded by the nucleic acid molecule is at least about 50% to about 60%, more preferably at least about 60% to about 70%, more preferably at least about 70% , More preferably about 70% to 80%, 80% to 90%, 90% to 95%, and most preferably about 96%, 97%, 98% % Or 99% or more homologous.
[84] Sequences are aligned for optimal comparison purposes to determine two amino acid sequences (e.g., one of the amino acid sequences of the invention and variant forms thereof) or the homology (%) of the two nucleic acid sequences (e.g., A gap can be introduced into the sequence of one protein or nucleic acid for optimal alignment with other proteins or nucleic acids). The amino acid residue or nucleotide corresponding to the amino acid position or the nucleotide position is then compared. When a position within one sequence (e.g., one of the amino acid sequences of the invention) is occupied by the same amino acid residue or nucleotide as the corresponding position within another sequence (e.g., variant form of the amino acid sequence) (I. E., &Quot; homology " of an amino acid or nucleic acid, as used herein, is equivalent to the " identity " of an amino acid or nucleic acid). Homology (%) between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology (% = number of identical positions / total number of positions x 100).
[85] Addition or deletion of one or more nucleotides in the nucleotide sequence of the present invention so that the substitution, addition or deletion of one or more amino acids is introduced into the encoded protein, the protein sequence of the present invention (e. G., The even sequence of the sequence listing Lt; / RTI > sequence of SEQ ID NO: 1). Mutations can be introduced into one of the nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions can be made at one or more expected non-essential amino acid residues. &Quot; Conserved amino acid substitution " refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are defined in the art. These groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, (Alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) having a beta-branched side chain, amino acids having a beta-branched side chain (for example, asparagine, glutamine, serine, threonine, tyrosine, cysteine) (E.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the nonessential amino acid residues predicted in the PTS protein are preferably substituted with other amino acid residues from the same side chain group. Alternatively, in another embodiment, the mutation can be randomly introduced along all or part of the PTS coding sequence, for example, by a saturation mutagenesis method, and can be generated Can be screened for the PTS activity described herein. One nucleotide sequence of the odd-numbered sequences of the sequence listing can be mutagenized, and then the encoded protein can be recombinantly expressed, and the activity of the protein can be determined, for example, using the assays described herein See Example 8).
[86] In addition to nucleic acid molecules encoding the PTS proteins described above, another aspect of the invention relates to isolated nucleic acid molecules that are antisense thereto. An " antisense " nucleic acid comprises a nucleotide sequence complementary to a " sense " nucleic acid encoding a protein, for example, complementary to a coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Thus, the antisense nucleic acid can be hydrogen bonded to the sense nucleic acid. The antisense nucleic acid may be complementary only to the entire PTS coding strand or a portion thereof. In one embodiment, the antisense nucleic acid molecule is antisense to the " coding region " of the coding strand of the nucleotide sequence encoding the PTS protein. The term " coding region " means a region of the nucleotide sequence comprising a codon translated into an amino acid residue (e.g., the entire coding region of SEQ ID NO: 5 (RXA01503) includes nucleotides 1 to 249). In another embodiment, the antisense nucleic acid molecule is antisense to the " noncoding region " of the coding strand of the nucleotide sequence encoding the PTS. The term " noncoding region " means 5 ' and 3 ' sequences (i.e., 5 ' and 3 ' non-translated regions) on both sides of the coding region that are not translated into amino acids.
[87] Based on the coding strand sequence coding for the PTS disclosed herein (for example, this sequence is described in odd numbered sequence numbers in the sequence listing), according to the base pairing rules of Watson and Crick, Of antisense nucleic acid can be designed. The antisense nucleic acid molecule may be complementary to the entire coding region of the PTS mRNA, but more preferably is an oligonucleotide that is only antisense to a portion of the coding or noncoding region of the PTS mRNA. For example, the antisense oligonucleotide may be complementary to the region around the translation initiation site of the PTS mRNA. Antisense oligonucleotides are, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleic acid of the present invention can be produced using a chemical synthesis method and an enzymatic ligation reaction method using a method known in the art. For example, a variety of modified nucleotides designed to increase the biological stability of a naturally occurring nucleotide, or a molecule, or to increase the physical stability of a double strand formed between the antisense and sense nucleic acids (e. Phosphorothioate derivatives and acridine substituted nucleotides may be used), antisense nucleic acids (e.g., antisense oligonucleotides) may be chemically synthesized. Examples of modified nucleotides that can be used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- (Carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl uracil, dihydrouracil, beta-D-galactosylquosine, inosine, N6-isopentenyl adenine Methylguanidine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl Aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl Adenine, uracil-5-oxyacetic acid (v), bibutoxosine, pseudouracil, quiosine, 2-thiocytosine, 5-oxyacetic acid, methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- -3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, an expression vector in which the nucleic acid is subcloned in the antisense orientation (i.e., the RNA transcribed from the inserted nucleic acid is antisense orientation to the target nucleic acid of interest, as further described in the following subsection) .
[88] The antisense nucleic acid molecules of the present invention are typically administered to cells or they hybridize with, or bind to, mRNA and / or genomic DNA of cells encoding the PTS protein, for example by inhibiting transcription and / or translation , And is produced in the reaction system to suppress expression of the protein. Hybridization may be by conventional nucleotide complementarity to form stable duplexes or by the case of antisense nucleic acid molecules that bind to DNA double strands through specific interactions, for example in a major groove of double helix . The antisense molecule can be modified to bind specifically to a receptor or antigen expressed on the selected cell surface, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. The vector described herein may also be used to deliver an antisense nucleic acid molecule to a cell. In order to achieve a sufficient intracellular concentration of the antisense molecule, a vector construct in which the antisense nucleic acid molecule lies under the control of a strong prokaryotic, viral or eukaryotic cell promoter is preferred.
[89] In another embodiment, the antisense nucleic acid molecule of the present invention is an a-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with a complementary RNA, in contrast to the usual β-units, the strands are parallel to each other (Gaultier et al . (1987) Nucleic Acids. Res . 15: 6625-6641). The antisense nucleic acid molecule is a 2'-o-methyl ribonucleotide [Inoue et al . (1987) Nucleic Acids Res. 15: 6131-6148] or a chimeric RNA-DNA analogue [Inoue et al . (1987) FEBS Lett . 215: 327-330).
[90] In another embodiment, the antisense nucleic acid molecule of the invention is a ribozyme. The ribozyme is a catalytic RNA molecule having ribonuclease activity and can cleave a single-stranded nucleic acid such as an mRNA having a complementary region thereto. Thus, a PTS mRNA transcript was transiently transfected with a catalytic (e. G., Transfected) transfectant using a ribozyme (e.g., as described in Hasmerhoff and Gerlach (1988) Nature 334: 585-591) The ribozyme having specificity for the PTS-encoding nucleic acid can be designed based on the nucleotide sequence of the PTS DNA disclosed herein (i.e., SEQ ID NO: 5 (RXA01503)). For example, a derivative of Tetrahymena L-19 IVS RNA can be made wherein the nucleotide sequence of the active site is complementary to the nucleotide sequence cleaved in the PTS-coding mRNA (see, for example, US Patent No. 4,987,071 to Cech et al. And U.S. Patent No. 5,116,742 to Cech et al.) Alternatively, PTS mRNA can be used to screen for catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules I can do it (See, for example, Bartel, D. and Szostak, JW (1993) Science 261: 1411-1418).
[91] Alternatively, expression of the PTS gene can be achieved by targeting a complementary nucleotide sequence to the regulatory region of the PTS nucleotide sequence (e.g., the PTS promoter and / or enhancer) to form a triple helix structure that prevents transcription of the PTS gene in the target cell 6 (6): 569-84; Helene, C. et al. (1992) Ann. NY Acad. Sci. 660: 27-36; and Maher, LJ (1992) Bioassays 14 (12): 807-15).
[92] B. Recombinant expression vectors and host cells
[93] Another aspect of the invention relates to an expression vector comprising a nucleic acid encoding a vector, preferably a PTS protein (or portion thereof). As used herein, the term " vector " means a nucleic acid capable of transporting another nucleic acid linked thereto. One type of vector, " plasmid " refers to a circular double-stranded DNA ring to which additional DNA fragments can be ligated. Other types of vectors, viral vectors, can ligate additional DNA fragments into the viral genome. Certain vectors are capable of self-replication within introduced host cells (e. G. Bacterial vectors with bacterial cloning start points and mammalian episome vectors). Other vectors (e. G., Non-episomal vectors of mammals) are inserted into the host genome at the time of introduction into the host cell and thereby replicated along with the host genome. In addition, certain vectors may direct expression of operably linked genes. Such a vector is referred to herein as an " expression vector. &Quot; Generally, expression vectors used in recombinant DNA techniques usually have the form of a plasmid. Since the plasmid is the most commonly used vector form, the term " plasmid " and " vector " can be used in combination. The present invention also includes other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that provide equivalent functions.
[94] The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in the host cell, and a suitable form is one in which the recombinant expression vector contains one or more regulatory sequences selected on the basis of the expression host cell ≪ / RTI > operatively linked to a sequence). Within a recombinant expression vector, " operably linked " means that the desired nucleotide sequence is linked to the regulatory sequence (s) to enable expression (e.g., in an in vitro transcription / translation system or when the vector is introduced into a host cell In host cells). The term " regulatory sequence " is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include constitutive expression of a nucleotide sequence within a plurality of host cell types and allowing expression of the nucleotide sequence only within a particular host cell. Preferred regulatory sequences include, for example cos- used preferably in bacteria, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI ^q -, T7-, T5 -, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, lambda-P _R- or lambda P _L. Additional regulatory sequences include, for example, promoters from yeast and fungi such as ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, CaMV / 35S, SSU, OCS, lib4, , B33, nos, ubiquitin- or tracer-promoter. Artificial promoters can also be used. Those skilled in the art will appreciate that the expression vector design may vary depending on factors such as the choice of host cell to be transformed, the degree of expression of the desired protein, and the like. The expression vector of the present invention can be introduced into host cells to produce proteins or peptides including fusion proteins or peptides encoded by the nucleic acids described herein (e.g., PTS proteins, mutants of PTS proteins, fusion proteins, etc.) .
[95] The recombinant expression vector of the present invention can be designed for expression of PTS protein in prokaryotic or eukaryotic cells. For example, the PTS gene may be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (Romanos, MA et al . yeast: a review. ", Yeast 8:. 423-488; van den Hondel, CAMJJ et al (1991)" Heterologous gene expression in filamentous fungi "in: More gene Manipulations in Fungi, JW Bennet & LL Lasure, eds, p (1991) " Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Pebery, JF et al ., ≪ RTI ID = 0.0 > eds., pp. 1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells (Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefactiens -mediated transformation of Arabidopsis thaliana leaf and cotyledon explants " Plant Cell Rep .: 583-586) or may be expressed in mammalian cells All.
[96] Suitable host cells are further described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerases.
[97] Expression of the protein in prokaryotic cells is generally carried out with a constitutive or inducible promoter-containing vector such that a fusion or non-fusion protein is expressed. The fusion vector adds a number of amino acids to the protein to be coded, generally to the amino terminus of the recombinant protein. Such fusion vectors are typically for three purposes: 1) increased expression of the recombinant protein, 2) increased solubility of the recombinant protein, and 3) ease of purification of the recombinant protein by acting as a ligand in affinity purification. Usually, in the fusion expression vector, a protein cleavage site is introduced at the junction of the fusion residue and the recombinant protein, and it is possible to purify the fusion protein and then to isolate the recombinant protein from the fusion residue. Such enzymes and their cognate recognition sequences include coagulation factor Xa, thrombin and enterokinase.
[98] Exemplary fusion expression vectors include pGEX (see Pharmacia Biotech Inc; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ), which respectively fuse the guluta thion-S-transferase (GST), maltose E binding protein or protein A (protein A) to the target recombinant protein. In one embodiment, the coding sequence of the PTS protein is cloned into a pGEX expression vector to generate a fusion protein coding vector containing the GST-thrombin cleavage site-X protein at the N-terminus to the C-terminus. The fusion protein can be purified by affinity chromatography using a Guluta Taion-agarose resin. Recombinant PTS proteins not fused to GST can be obtained by truncating the fusion protein into thrombin.
[99] Suitable inducible non-fusion. Examples of the vector of the present invention include pTrc (see Amann et al ., (1988) Gene 69: 301-315), pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pINIII113-B1, λgt11, pBdCl , and pET 11d (document [Studier et al, Gene Expression Technology :. Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89] and the method disclosed in [Pouwels et al ., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018). The expression of the target gene from pTrc is due to the transcription of the host's RNA polymerase through the hybrid trp-lac fusion promoter. Expression of the target gene from the pET 11d vector is by transcription mediated by the co-expressed viral RNA polymerase (T7 gn1) through the T7 gn 10-lac fusion promoter. This viral polymerase is supplied by host strain BL21 (DE3) or HMS174 (DE3) from parental [lambda] prophage containing the T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. An appropriate vector can be selected for transformation of various bacteria. For example, the plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful for transforming the Streptomyces genus, while the plasmids pUB110, pC194 or pBD214 are suitable for transforming bacillus species. Plasmids used to transfer genetic information into Corynebacterium include pHM1519, pBL1, pSA77 or pAJ667 (see Pouwels et al ., Eds. (1985) Cloning Vectors.Elsevier: New York IBSN 0 444 904018) do.
[100] One strategy to maximize the expression of recombinant proteins is to express the protein in host bacteria that reduces the ability to cleave the recombinant protein by proteolytic cleavage (Gottesman, S., Gene Expression Technology: Methodsin Enzymology 185 , Academic Press, San Diego, California (1990) 119-128). Another strategy is to modify the nucleic acid sequence of the nucleic acid inserted into the expression vector so that the individual codons for each amino acid are the preferred codons for the bacteria selected for expression (e.g., cglutamicum) [Wada et al . (1992) Nucleic Acids Res . 20: 2111-2118]. As such, modification of the nucleic acid sequence of the present invention can be performed by standard DNA synthesis techniques.
[101] In another embodiment, the PTS protein expression vector is a yeast expression vector. Yeast s. Examples of expression vectors of Serrebia are pYepSec1 (Baldari, et al ., (1987) Embo J. 6: 229-234), 2μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa Herskowitz, (1982) Cell 30: see 933-943)), pJRY88 (literature [Shultz et al, (1987) Gene 54:. 113-123] reference) and include Invitrogen Corporation, San Diego, CA ( pYES2) . Methods for preparing vectors and vectors suitable for other fungi such as filamentous fungi include van den Hondel, CAMJJ & Punt, PJ (1991) Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, ... JF Peberdy, et al , eds, p 1-28, Cambridge University Press: Cambridge] , and the literature [Pouwels et al, eds (1985 ) Cloning Vectors Elsevier:... in New York (IBSN 0 444 904018) ] The above-mentioned ones are included.
[102] Alternatively, baculovirus expression vectors can be used to express the PTS proteins of the invention in insect cells. Baculovirus vectors for protein expression in cultured insect cells (e.g., Sf 9 cells) include the pAc system (Smith et al . (1983) Mol . Cell Biol . 3: 2156-2165) See Lucklow and Summers (1989) Virology 170: 31-39).
[103] In another embodiment, the PTS protein of the invention can be expressed in plant cells from single cell plant cells (e. G., Algae) or higher plants (e. G., Seed plants such as crop plants). Examples of plant expression vectors include those described in Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol . 20: 1195-1197, Bevan, MW (1984) " Binary Agrobacterium vectors for plant transformation ", Nucl. Acid. Res . 12: 8711-8721) and include pLGV23, pGHlac +, pBIN19, pAK2004 and pDH51 (see Pouwels et al ., Eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018) do.
[104] In another embodiment, a mammalian expression vector is used to express the nucleic acid of the invention in mammalian cells. Examples of mammalian expression vectors include pCDM8 (see Seed, B. (1987) Nature 329: 840) and pMT2PC (see Kaufman et al . (1987) EMBO J. 6: 187-195) . When using mammalian cells, the regulatory function of the expression vector is usually by a viral regulatory element. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. Other expression systems suitable for both prokaryotic and eukaryotic cells are described in Chapters 16 and 17 of Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd. ed. Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
[105] In another embodiment, the recombinant mammalian expression vector allows differential nucleic acid expression in a particular cell type (e.g., a tissue-specific modulating element is used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Specific examples of suitable tissue-specific promoters include, but are not limited to, albumin promoters (liver-specific; see Pinkert et al . (1987) Genes Dev . 1: 268-277), lymphoid-specific promoters [Calame and Eaton (1988) Adv Immunol 43:.. 235-275] reference), in particular T-cell promoter (as described in [Winoto and Baltimore (1989) EMBO J 8.: 729-733] of the reference receptor) and an immunoglobulin (See for example, Banerji et al . (1983) Cell 33: 729-740], Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g., neurofilament promoters; Specific promoters (see Edlund et al . (1985) Science 230: 912-916) and mammary-specific (see Byrne and Ruddle (1989) PNAS 86: 5473-5477) Promoters (e. G., Whey promoters; see U.S. Pat. No. 4,873,316 and European Application No. 264,166). In addition, development-regulated promoters such as the rat hox promoter (see Kessel and Gruss (1990) Science 249: 374-379) and the alpha -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev . 3: 537-546).
[106] The present invention further provides a recombinant expression vector containing the DNA molecule of the invention cloned in an expression vector in the antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence such that expression of the RNA molecule that is antisense to the PTS mRNA (by transcription of the DNA molecule) is operable. Regulatory sequences operably linked to the nucleic acid cloned in the antisense orientation can be selected to allow the constant expression of the antisense RNA molecule in various cell types, such as, for example, viral promoters and / or enhancers, , Tissue-specific, or cell-type specific expression. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus that produces an antisense nucleic acid under the control of a highly efficient regulatory region, the activity of which may be determined by the cell type into which the vector is introduced. For controlling gene expression using antisense genes, see Weintraub, H. et al ., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics , Vol. 1 (1) 1986).
[107] Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms " host cell " and " recombinant host cell " are used interchangeably herein. Such terms are understood to refer not only to a particular subject cell but also to a progeny or potential progeny of such a cell. In fact, such offspring are not identical to the parent cell, but are still included in the scope of the term as used herein, since any variation may occur due to mutations or environmental influences over the generations.
[108] The host cell may be any prokaryotic or eukaryotic cell. For example, the PTS protein is Mr. Insect cells, yeast or mammalian cells (e. G., Chinese rat ovary cells (CHO) or COS cells) such as glutamicum. Other suitable host cells are known to those skilled in the art. Table 3 lists the Corynebacterium glutamicum-related microorganisms that can be readily used as host cells for the nucleic acid and protein molecules of the present invention.
[109] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms " transformation " and " transfection " refer to a nucleic acid in the form of a heterologous nucleic acid (eg, a linear DNA or RNA (eg, a single gene construct that is not a vector, Plasmid, phage, parasite, phagemid, transposon, or other DNA) into the host cell, and it is also possible to use calcium phosphate or calcium chloride simultaneous precipitation method, DEAE-dextran-light oil transfusion method, Includes lipofection or electroporation. Suitable methods for transforming or transfecting host cells are described in Sambrook et al . ( Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)] and other experimental guidelines.
[110] For stable transfection of mammalian cells, it is known that, according to the expression vector and the transfection technique used, only a few hydrophobic cells insert heterologous DNA into its genome. To identify and select these inserts, the gene encoding the selectable marker (e.g., antibiotic resistance) is generally introduced into the host cell along with the desired gene. Preferred selectable markers include those that confer drug resistance such as G418, hygromycin and methotrexate. The nucleic acid encoding the selectable marker may be introduced into the host cell on the vector encoding the PTS protein, or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug screening (eg, cells with the selectable marker gene will survive, but other cells will die).
[111] To prepare homologous recombinant microorganisms, deletion, addition, or substitution is introduced to produce a vector containing at least a portion of the PTS gene that has been modified (e.g., disrupted) by the PTS gene. Preferably, the PTS gene is a Corynebacterium glutamicum PTS gene, but may be a homologue from a related bacterial as well as a mammalian, yeast or insect origin. In a preferred embodiment, the vector is designed such that the endogenous PTS gene is functionally disrupted by homologous recombination (i. E., Also referred to as a " knock-out " vector that no longer encodes a functional protein). Alternatively, the vector is designed such that the endogenous PTS gene is mutated or otherwise modified by homologous recombination, but still encoding a functional protein (i.e., the upstream regulatory region may be modified to alter the expression of the endogenous PTS protein). The homologous recombination vector contains additional nucleic acid of the PTS gene on the 5 'and 3' side of the modified PTS gene fragment, allowing homologous recombination between the exogenous PTS gene carried by the vector and the endogenous PTS gene in the microorganism. The added side PTS nucleic acid has a length sufficient to successfully homologous recombine with the endogenous gene. Typically, several kilobases of side DNA (both 5 'and 3' ends) are contained within the vector (see, for example, Thomas, KR, and Capecchi, MR (1987) Cell 51: 503). The vector is introduced into the microorganism (e. G., By electroporation), and the homologous recombination of the introduced PTS gene and the endogenous PTS gene is selected by known techniques.
[112] In another embodiment, the recombinant microorganism can be prepared by including a screening system to regulate the expression of the introduced gene. For example, if the PTS gene is located in the vector under the control of the lac operon, expression of the PTS gene is possible only in the presence of IPTG. Such regulatory systems are well known in the art.
[113] In another embodiment, the endogenous PTS gene in the host cell is destroyed (e. G., By homologous recombination or other genetic means known in the art) so that expression of its protein product does not occur. In another embodiment, a PTS gene introduced into an endogenous or host cell is modified by one or more point mutations, deletions or inversions, but still encodes a functional PTS protein. In another embodiment, one or more regulatory regions (e. G., Promoters, repressors, or inducers) in a microorganism are modified (e. G., By deletion, truncation, inversion, or point mutation) to alter the expression of the PTS protein. Those skilled in the art will appreciate that host cells containing one or more of the PTS genes and protein modifications described above can be readily prepared using the methods of the present invention and are included in the present invention.
[114] A host cell of the invention, such as a cultured prokaryotic or eukaryotic host cell, produces (i.e., expresses) a PTS protein. Accordingly, the present invention further provides a method for producing PTS protein using the host cell of the present invention. In one embodiment, the method comprises administering a host cell of the invention (wherein a recombinant expression vector encoding a PTS protein is introduced, or a gene encoding a wild-type or modified PTS protein is introduced into the genome) until the PTS protein is produced And culturing in a suitable medium. In another embodiment, the method further comprises isolating the PTS protein from the medium or host cell.
[115] C. Isolated PTS protein
[116] Another aspect of the invention relates to isolated PTS proteins and biologically active fragments thereof. An "isolated" or "purified" protein or biologically active fragment thereof is substantially free of cellular material when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized . The term " substantially free of cellular material " includes a PTS protein preparation in which the protein is isolated from the cellular constituents of cells that produce the PTS protein, either naturally or by recombination. In one embodiment, the term " substantially free of cellular material " means that the non-PTS protein (also referred to herein as "contaminating protein") content is less than about 30% (dry weight), preferably less than about 20% , More preferably less than about 10%, and most preferably about 5%. It is also preferred that when the PTS protein or biologically active fragment thereof is produced by recombination, there is substantially no culture medium. That is, a protein preparation in which the culture medium content is less than about 20% by volume, more preferably less than about 10% by volume, and most preferably less than 5% by volume is preferable. The term " substantially free of chemical precursors or other chemicals " includes a PTS protein preparation that separates the protein from chemical precursors or other chemicals involved in the synthesis of the PTS protein. In one embodiment, the term " substantially free of chemical precursors or other chemicals " means that the content of chemical precursors or non-PTS chemicals is less than about 30% (dry weight), preferably less than about 20% , ≪ / RTI > less than about 10%, and most preferably about 5%. In a preferred embodiment, the isolated protein or biologically active fragment thereof is free of contaminating proteins from the organism from which the PTS protein is derived. Typically, such proteins are, for example, seeds. Seeds in microorganisms such as glutamicum. And is produced by recombinant expression of the glutamicum PTS protein.
[117] The isolated PTS protein or fragment thereof of the present invention may participate in the transport of high energy carbon-containing molecules such as glucose into the C. glutamicum and also participate in intracellular signaling within the microorganism, May have more than one activity. In a preferred embodiment, the protein or fragment thereof comprises an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (e. G., The sequence is even in SEQ ID NO: 1 in the Sequence Listing) and is a seed of a high energy carbon containing molecule such as glucose. To participate in the transport into glutamicum, or to maintain the ability to participate in intracellular signaling within this microorganism. The fragment of the protein is preferably a biologically active fragment as described herein. In another preferred embodiment, the PTS protein of the present invention has an amino acid sequence as set forth in the Sequence Listing, wherein the sequence number is even in the Sequence Listing. In another preferred embodiment, the PTS protein has an amino acid sequence that is encoded by a nucleotide sequence that hybridizes (e. G., Hybridizes under stringent conditions) to the nucleotide sequence of the invention (e. G., The sequence SEQ ID NO: . In another preferred embodiment, the PTS protein has a homology of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% 58%, 59% or 60%, preferably about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , More preferably at least 95%, 96%, 97%, 98%, 99% or more nucleotides, preferably at least 95%, 88%, 89% or 90% or 91%, 92%, 93%, 94% Lt; RTI ID = 0.0 > sequence. ≪ / RTI > Also, ranges of identity values (e.g., 70-90% identity or 80-95% identity) that are intermediate values to the above-mentioned values are included in the present invention. For example, ranges of identity values using any of the above-mentioned values as a combination of a maximum value and a minimum value are included in the present invention. Also preferred PTS proteins of the invention preferably possess one or more of the PTS activities described herein. For example, a preferred PTS protein of the present invention comprises an amino acid sequence encoded by a nucleotide sequence that hybridizes (e.g., hybridizes under stringent conditions) to the nucleotide sequence of the present invention, and comprises a seed of a high energy carbon-containing molecule such as glucose. May participate in the transport into glutamicum and also participate in intracellular signaling within this microorganism, or may have one or more of the activities set forth in Table 1.
[118] In another embodiment, the PTS protein is substantially homologous to the amino acid sequence of the invention (e. G., The sequence with the even sequence number in the Sequence Listing), and because of natural mutations or mutations as described above in subsection I, But also retains the functional activity of one of the amino acid sequences of the present invention. Thus, in another embodiment, the PTS protein has homology to the entire amino acid sequence of the invention of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% % Or 60% or more, preferably about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% or more, more preferably about 71% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 89% or 90%, or 91%, 92%, 93%, 94% or more, and more preferably about 95%, 96%, 97%, 98%, 99% , And one or more of the PTS activities described herein. Also, ranges of identity values (e.g., 70-90% identity or 80-95% identity) that are intermediate values to the above-mentioned values are included in the present invention. For example, ranges of identity values using any of the above-mentioned values as a combination of a maximum value and a minimum value are included in the present invention. In another embodiment, the invention is directed to a full length C. Glutamicum protein that is substantially homologous to the entire amino acid sequence of the invention.
[119] The biologically active fragment of the PTS protein comprises an amino acid sequence derived from the amino acid sequence of the PTS protein (e. G., Sequence with an even numbered sequence in the Sequence Listing) or comprises the amino acid sequence of a protein homologous to the PTS protein, Or less than the full-length protein that is homologous to the PTS protein and exhibits one or more PTS protein activities. Typically, biologically active fragments (peptides, e.g., peptides having amino acid lengths of 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, Domains or motifs having at least one activity of the protein. In addition, fragments with other regions of the protein deleted and having biologically different activities can be produced by recombinant techniques and one or more activities described herein can be determined. Preferably, the biologically active fragment of the PTS protein comprises one or more selected domains / motifs or fragments thereof having biological activity.
[120] The PTS protein is preferably produced by recombinant DNA technology. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above), and the PTS protein is introduced into the host cell Lt; / RTI > The PTS protein can then be isolated from the cell through a suitable purification step using standard protein purification techniques. Aside from recombinant expression, PTS proteins, polypeptides or peptides can be chemically synthesized using standard peptide synthesis techniques. In addition, native PTS proteins can be isolated from cells (e. G., Endothelial cells) using, for example, anti-PTS antibodies that can be produced by standard methods using the PTS proteins or fragments thereof of the invention.
[121] The invention also provides a PTS chimeric or fusion protein. As used herein, a PTS " chimeric protein " or " fusion protein " comprises a PTS polypeptide operably linked to a non-PTS polypeptide. &Quot; PTS polypeptide " refers to a polypeptide having an amino acid sequence corresponding to a PTS, wherein the term " non-PTS polypeptide " refers to a protein that is substantially different from a protein that is not homologous to the PTS protein, Quot; means a polypeptide having an amino acid sequence corresponding to a protein. In a fusion protein, the term " operably linked " means that the PTS and non-PTS polypeptides are fused together within a frame. The non-PTS polypeptide may be fused to the N-terminus or C-terminus of the PTS polypeptide. For example, in one embodiment, the fusion protein is a GST-PTS fusion protein in which the PTS sequence is fused to the C-terminus of the GST sequence. Such fusion proteins can facilitate the purification of recombinant PTS proteins. In another embodiment, the fusion protein is a PTS protein that contains a heterologous signal sequence at its N-terminus. Within a particular host cell (e.g., a mammalian host cell), the expression and / or secretion of the PTS protein may be increased by using heterologous signal sequences.
[122] Preferably, the PTS chimeric or fusion proteins of the invention are produced by standard recombinant DNA techniques. For example, DNA fragments encoding distinct polypeptide sequences may be treated according to conventional techniques, for example using blunt-ended or crossed ends for ligation, treating the restriction enzymes to generate appropriate ends, , The complementary ends are filled and the alkaline phosphatase is treated to avoid undesirable linkage and ligated together in the same frame by ligation with the enzyme. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of a gene fragment can be performed using a fixed primer, which causes a complementary overhang between two consecutive gene fragments, which is subsequently annealed and reamplified to generate a chimeric gene sequence (See, for example, Current Protocols in Molecular Biology, eds. Ausubel et al ., John Wiley & In addition, many expression vectors that previously encode fusion residues (e.g., GST polypeptides) are commercially available. The PTS-encoding nucleic acid may be cloned into such an expression vector such that the fusion residues are linked in the same frame to the PTS protein.
[123] The homologue of the PTS protein can be produced by mutation, for example, by fractionation mutation or by truncation of the PTS protein. As used herein, " homologue " refers to a variant form of the PTS protein that acts as an agonist or antagonist to the activity of the PTS protein. The agonist of the PTS protein may have substantially the same or a similar biological activity as the PTS protein. An antagonist of a PTS protein may inhibit one or more activities of a native PTS protein by competitively binding to a downstream or upstream member of the PTS system, including, for example, a PTS protein. Therefore, The glutamicum PTS protein and its homologues can modulate one or more sugar transport pathways or intracellular signaling pathway pathways in which the PTS protein plays a role in this microorganism.
[124] In another embodiment, the homologue of the PTS protein can be identified by screening a combinatorial library of mutations of the PTS protein (e.g., a mutation of the tRNA) against the PTS protein agonist or antagonist activity. In one embodiment, a library in which PTS variants are mixed can be obtained, for example, by ligation of the synthesized oligonucleotide mixture to the gene sequence with an enzyme so that the fused set of potential PTS sequences can be expressed as individual polypeptides, Such as a large fusion protein containing a set of sequences (e. G. Phage display). There are a variety of methods available for producing libraries of potential PTS homologues from foster oligonucleotide sequences. Chemical synthesis of the full-length gene sequence can be performed in an automated DNA synthesizer, and then the synthetic gene can be ligated into an appropriate expression vector. A set of gene scaffolds can be used to store all sequences encoding a desired set of potential PTS sequences within a mixture. Methods for synthesizing dimeric oligonucleotides are known in the art (see, for example, Narang, SA (1983) Tetrahedron 39: 3, Itakura et al . (1984) Annu. Rev. Biochem. 53: 323) , literature reference) [Itakura et al (1983) Science 198:.: 1056], reference [477 Ike et al (1983) Nucleic Acid Res 11..].
[125] In addition, a fragment library of PTS protein coding genes can be used to screen a homolog of the PTS protein and generate a mixed population of PTS fragments for subsequent screening. In one embodiment, the nuclease is treated with a double-stranded PCR fragment of the PTS coding sequence under the condition that the nick occurs about once per molecule, the double-stranded DNA is denatured, and the sense / antisense Degeneration of the DNA to form double-stranded DNA that may comprise a single nucleotide of the double-stranded DNA sequence, treatment of the S nuclease to remove single-stranded portions from the reconstructed double strand, and ligation of the resulting fragment library into an expression vector, A library of fragments can be prepared. By this method, expression libraries can be derived that encode N-terminal, C-terminal and internal fragments of PTS proteins of varying sizes.
[126] A number of techniques are known in the art for screening for gene products in combinatorial libraries made by point mutations or truncation and screening for gene products with properties selected in cDNA libraries. Such techniques can be used to quickly screen gene libraries generated by combination mutagenesis of PTS homologues. As the most widely used techniques, techniques available for high throughput analysis for screening large gene libraries are typically those in which a gene library is cloned into a replicable expression vector, the resulting vector is transformed into a suitable cell, Detection of the activity includes expression of the combination gene under conditions that facilitate isolation of the vector encoding the gene product for which detection is made. Recursive ensemble mutagenesis (REM), a novel technique for increasing the frequency of functional mutations in libraries, can be used in combination with screening assays to identify PTS homologs (Arkin and Yourvan, 1992 ) PNAS 89: 7811-7815, Delgrave et al . (1993) Protein Engineering 6 (3): 327-331).
[127] D. Uses and Inventions of the Invention
[128] The nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors, and host cells described herein may be used to identify Corynebacterium glutamicum and related organisms, genome mapping of organisms associated with Corynebacterium glutamicum, Identification and location of target sequences of Corynebacterium glutamicum, evolution studies, determination of PTS protein sites required for function, regulation of PTS protein activity, regulation of PTS pathway activity, and cell production of desired compounds such as fine chemicals And the like.
[129] The PTS nucleic acid molecules of the present invention have a variety of uses. First, it can be used to identify the presence of Corynebacterium glutamicum or its close homologues. It can also be used to confirm the presence of Corynebacterium glutamicum or its close homologues in the mixed microorganism group. The present invention relates to a method for detecting the presence or absence of the above-mentioned organism by using probes unique to Corynebacterium glutamicum and probing the genomic region to probe a genomic DNA extracted from a culture of a single or mixed microorganism group under stringent conditions, Provides the nucleic acid sequence of the four bacterial glutamicum genes.
[130] Corynebacterium glutamicum is inherently non-pathogenic, but is associated with Corynebacterium diphtheriae . Corynebacterium diphtheria is a causative organism of diphtheria, a rapidly progressive acute febrile infection with local and systemic lesions. In these diseases, local lesions develop on the upper part of the respiratory tract, necrotic damage to the epithelium, and Bacillus secrete toxins and spread to tissues susceptible to infection at the end of the body through the lesions. Degenerative changes caused by inhibition of protein synthesis in the tissues, including the heart, muscle peripheral nerves, adrenal glands, kidney, liver and spleen, cause systemic pathology of the disease. Diphtheria maintains high incidence in many parts of the world, including in Africa, Asia, Eastern Europe and the former Soviet Union. Continued pandemic diphtheria in Eastern Europe and the former Soviet Union has resulted in more than 5,000 deaths since 1990.
[131] In one embodiment, the invention provides a method of identifying the presence or activity of Corynebacterium diphtheria in an individual. The present invention includes detecting the presence or activity of Corynebacterium diphtheria in an individual by detecting at least one nucleic acid or amino acid sequence of the invention in the subject (represented by an odd or even sequence number in the sequence listing, respectively). Corynebacterium glutamicum and Corynebacterium diphtheria are related bacteria, and many nucleic acid and protein molecules of Corynebacterium glutamicum have homology with nucleic acid and protein molecules of Corynebacterium diphtheriae And can be used to detect Corynebacterium difterieri in an individual.
[132] In addition, the nucleic acid and protein molecules of the invention serve as markers for specific regions of the genome. This function is useful not only for genome mapping but also for functional studies of Corynebacterium glutamicum proteins. For example, the genome of the Corynebacterium glutamicum genome can be identified by cutting the genome of the Corynebacterium glutamicum and incubating the fragment with a DNA binding protein to bind a specific DNA binding protein of Corynebacterium glutamicum. The region that binds to the protein may also be probed with a nucleic acid molecule of the present invention, preferably a readily detectable label. The binding of the nucleic acid molecule to such a genomic fragment allows the position of the fragment to be located in the genome map of Corynebacterium glutamicum, and the nucleic acid sequence to which the protein binds can be quickly and easily determined when the enzyme is repeatedly carried out with different enzymes . In addition, the nucleic acid molecule of the present invention can have sufficient homology with the sequence of the associated species and can be used as a marker for creating genomic maps of related bacteria such as Brevibacterium lactofermentum .
[133] In addition, the PTS nucleic acid molecule of the present invention is useful for evolutionary and protein structure studies. The sugar-entry system involved by the molecules of the present invention is utilized by a wide variety of bacteria. The evolutionary association of an organism can be evaluated by comparing the sequence of a nucleic acid molecule of the present invention with a nucleic acid molecule sequence encoding an enzyme similar to another organism. Similarly, these comparisons can assess the conservation of sequence regions, and these conserved regions can help determine protein regions essential for enzyme function. This method of determination is useful in protein engineering and can indicate resistance to mutations induced without loss of protein function.
[134] By manipulating the PTS nucleic acid molecule of the present invention, a PTS protein having a function different from that of the wild-type PTS protein can be produced. Such a protein may have improved efficiency or activity, may be present in excess of normal cells, or may have decreased efficiency or activity.
[135] The present invention provides a method for screening molecules that modulate the activity of a PTS protein by interacting with the protein itself or a binding partner of a substrate or PTS protein, or by modulating the transcription or translation of a PTS nucleic acid molecule of the invention. In this method, an organism expressing one or more PTS proteins of the invention is contacted with one or more test compounds to assess the effect of each test compound on the activity or expression level of the PTS protein.
[136] The PTS molecules of the present invention can be modified to improve the yield, yield and / or production efficiency of one or more fine chemicals. For example, by modifying the PTS protein involved in glucose entry to optimize its activity, the rate of glucose flux or the rate at which glucose moves into the cell can be increased. The degradation of intracellular glucose and other sugars provides the energy that can be used to cause biochemical reactions that are unlikely to occur energetically, such as those involved in the biosynthesis of fine chemicals. Such degradation also provides intermediates and precursors necessary for the biosynthesis of certain fine chemicals such as amino acids, vitamins and cofactors. By increasing the amount of intracellular high energy carbon molecules through modification of the PTS molecules of the present invention, both the energy that can be used to carry out the metabolic pathway required for one or more fine chemicals and the intracellular pool of metabolites necessary for such production are both increased . Conversely, by reducing the uptake of sugars which contain as a degradation product a compound used solely in the metabolic pathway competing with the pathway for production of the desired fine chemicals for enzymes, cofactors or intermediates, the pathway is reduced and regulated accordingly It would be possible to increase production through the target biosynthetic pathway.
[137] Further, the PTS molecule of the present invention is a seed. Can participate in one or more intracellular signaling pathways that can affect the yield and / or production rate of one or more fine chemicals in glutamicum. For example, proteins (e.g., members of the HPr, the Enzyme I, or the Enzyme II complex) that are required for the introduction of one or more sugars from the extracellular medium can be produced in a post-translational process It can be deformed. Examples of this phenomenon include: In E. coli, fructose 1,6-bisphosphate exhibits a high intracellular concentration, which phosphorylates serine 46 of HPr such that the molecule is no longer involved in the inflow of any sugars. The intracellular concentration of the sugar in which the entry system is interrupted may be sufficient to maintain the normal function of the cell, but the overproduction of the intended fine chemicals may be limited. Thus, by preventing the PTS protein of the present invention from further reacting to such negative regulation, higher intracellular concentrations of one or more sugars can be produced, and moreover, one or more fine chemicals from living organisms containing such mutant PTS proteins can be more efficiently It may be desirable to be able to produce at a higher yield.
[138] The method of inducing mutation that causes the PTS protein to increase the yield of the target compound is not limited, and modifications of these methods will be apparent to those skilled in the art. By these mechanisms, the nucleic acid and protein molecules of the present invention can be used to express mutant PTS nucleic acid and protein molecules. Glutamicum, or related strains to improve the yield, production and / or production rate of the desired compound. The desired compound is the intermediate product of the end product of the biosynthetic pathway and the natural metabolic pathway, In the glutamic acid metabolism, the seed of the present invention does not occur naturally. Seeds containing molecules produced by the Corynebacterium glutamicum strain. It can be all products produced by Corynebacterium glutamicum.
[139] The present invention is further illustrated by the following examples, but should not be construed as being limited thereto. All references, patent applications, patents, all content of published patent applications, tables and sequence listings cited herein are incorporated by reference.
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[161] Example 1: Preparation of whole genomic DNA of Corynebacterium glutamicum ATCC 13032
[162] Corynebacterium glutamicum (ATCC 13032) cultures in BHI medium (Difco product) were grown overnight at 30 ° C with vigorous shaking. The cells were recovered by centrifugation and the supernatant was discarded and the cells resuspended in 5 ml of buffer-I (5% of the initial volume of culture - all volumes indicated were calculated for 100 ml culture volume). The composition of Buffer-I was as follows: 140.34 g / L sucrose, 2.46 g / L MgS0 ₄ x 7 H ₂ O, 10 ml / L KH ₂ PO ₄ solution (100 g / the adjustment), 50 ml / L M12 concentrate _{(10 g / L (NH 4} ) 2 SO 4, 1 g / L NaCl, 2 g / L MgSO 4 × 7 H 2 O, 0.2 g / L CaCl 2, 0.5 g / L yeast extract (Difco product), 10 ml / L trace elements mixture _{(200 mg / L FeSO 4 ×} H 2 O, 10 mg / L ZnSO 4 × 7 H 2 O, 3 mg / L MnCl 2 × 4 H ₂ O, 30 mg / LH ₃ BO ₃ , 20 mg / L CoCl ₂ x 6 H ₂ O, 1 mg / L NiCl ₂ x 6 H ₂ O, 3 mg / L Na ₂ MoO ₄ x 2 H ₂ O, 500 L biotin, 0.2 mg / L folic acid, 20 mg / L p-aminobenzoic acid, 20 mg / L riboflavin, 40 mg / L ca. Lysozyme was added to the suspension to a final concentration of 2.5 mg / ml at 37 DEG C for approximately 4 hours, at a temperature of < RTI ID = 0.0 >≪ / RTI > followed by cell wall destruction and centrifugation The pellet was washed once with 5 ml Buffer-I and washed once with 5 ml TE-Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE 0.5 ml of SDS solution (10%) and 0.5 ml of NaCl solution (5 M) were added to the buffer. Proteinase K was added to a final concentration of 200 占 퐂 / ml and the suspension was incubated at 37 占 폚 Using a standard method, followed by extraction with phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol to purify the DNA. Next, 1/50 volume of 3 M sodium acetate and DNA was precipitated by the addition of 2 volumes of ethanol and then incubated at -20 DEG C for 30 minutes and centrifuged at 12,000 rpm for 30 minutes with a high speed centrifuge using an SS34 rotor (Sorvall product). The DNA was dissolved in 1 ml TE-Buffer containing 20 占 퐂 / ml RNaseA and dialyzed into 1000 ml TE-Buffer for 3 hours or more at 4 占 폚. During dialysis, the buffer was changed three times. 0.4 ml of dialyzed DNA solution was aliquoted, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol were added. After incubation at -20 ° C for 30 minutes, DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Hannover, Germany). The DNA pellet was dissolved in TE-buffer. The prepared DNA was used for all purposes, such as Southern blotting or genomic library production.
[163] Example 2: Genomic library of Corynebacterium glutamicum ATCC13032 in Escherichia coli
[164] (Sambrook, J. et al. (1989) " Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Laboratory (1989)) using the prepped DNA as described in Example 1 (See Ausubel, FM et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons).
[165] Any plasmid or cosmid could be used. In particular, plasmid pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA, 75: 3737-3741); (pBSSK +, pBSSK-, etc .; Stratagene, LaJolla, USA) or SuperCos I (Stratagene, La Jolla, USA), pCYC177 (Change & Cohen (1978) J. Bacteriol 134: 1141-1156) LaJolla, USA) or Lorist 6 (Gibson, TJ, Rosenthal A. and Waterson, RH (1987) Gene 53: 283-286). Specifically, a gene library for use in Corynebacterium glutamicum is prepared from the plasmid pSL 109 (Lee, H.-S. and AJ Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263) .
[166] Example 3 DNA Sequence Analysis and Computer Functional Analysis
[167] Using a genomic library as described in Example 2, DNA sequencing according to standard methods, in particular a chain termination method using an ABI377 sequencer (see, e.g., Fleischman, RD et al. (1995) Sequence analysis primers with the following nucleotide sequences were used: 5'-GGAAACAGTATGACCATG-3 '(SEQ ID NO: 1) (SEQ ID NO: 35) or 5'-GTAAAACGACGGCCAGT-3 '(SEQ ID NO: 36).
[168] Example 4: In vivo mutagenesis
[169] In vivo mutagenesis of Corynebacterium glutamicum may be carried out by using E. coli or other microorganisms with impaired ability to maintain complete genetic information (for example, Bacillus spp. Or Saccharomyces cerevisiae , etc.) (Or other vector) DNA in a yeast strain of the present invention. Exemplary mutagenic strains include, but are not limited to, genes related to DNA repair systems (e.g., mutHLS, mutD, mutT, etc .; see Rupp, WD (1996) DNA repair mechanisms in Escherichia coli and Salmonella , 2294, ASM: Washington). Such strains are known to those skilled in the art. The use of such strains is described, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.
[170] Example 5: DNA transfer between E. coli and Corynebacterium glutamicum
[171] Several Corynebacterium and Brevibacterium species contain endogenous plasmids autonomously replicating (such as pHM1519 or pBL1). (For a discussion of this, see, for example, Martin, JF et al. (1987) Biotechnology, 5: 137-146). The shuttle vector for use in Escherichia coli and Corynebacterium glutamicum is a standard vector used for Escherichia coli, containing a replication origin for Corynebacterium glutamicum and a suitable marker from Corynebacterium glutamicum Current Protocols in Molecular Biology ", John Wiley < RTI ID = 0.0 > (1994) " Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Laboratory Press, & Sons]). The origin of replication is preferably obtained from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. In particular, the gene used as a transformation marker for the above species is selected from the group consisting of a kanamycin resistance gene (for example, from Tn5 or Tn903 transposon) or a chloramphenicol resistance gene (Winnacker, EL (1987) to Gene Technology, VCH, Weinheim). There are numerous publications on how to make a wide variety of shuttle vectors that can be used for a variety of purposes, including replication in E. coli and Corynebacterium glutamicum, including gene overexpression (See, for example, Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin JF et al. (1987) Biotechnology, 5: 137-146 See Eikmanns, BJ et al. (1991) Gene, 102: 93-98).
[172] Using standard methods, the gene of interest can be cloned into one of the shuttle vectors described above, and such a hybrid vector can be introduced into Corynebacterium glutamicum strains. Transformation of Corynebacterium glutamicum can be carried out by the protoplast transformation method (Kastsumata, R. et al. (1984) J. Bacteriol. 159: 306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53: 399-303) and, when a particular vector is used, conjugation (see, for example, Schaefer, A et al. (1990) J. Bacteriol. -1666]). ≪ / RTI > In addition, plasmid DNA was prepared from Corynebacterium glutamicum by using preprep (using standard methods known in the art), transforming it with E. coli, and transferring a shuttle vector for use in Corynebacterium glutamicum to E. coli It is possible. Such a transformation step can be carried out using standard methods but it is advantageous to use a Mcr-deficient E. coli strain such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166: 1-19) Do.
[173] pCGl (US Pat. No. 4,617,267) or fragments thereof, and optionally a kanamycin resistance gene from TN903 (Grindley, ND and Joyce, CM (1980) Proc. Natl. Acad. ): 7176-7180) can be used to overexpress genes in Corynebacterium glutamicum strains. In addition, the plasmid pSL 109 (Lee, H.-S. and AJSinskey (1994) J. Microbiol. Biotechnol. 4: 256-263) was used to overexpress the gene in Corynebacterium glutamicum strains .
[174] In addition to using replicative plasmids, gene overexpression may also be achieved by insertion into the genome. Genomic insertion in Corynebacterium glutamicum or other Corynebacterium or Brevibacterium species can be achieved by homologous recombination with the genomic region (s), restriction endonuclease mediated insertion (REMI) [German Patent No. 19823834]), or using a transposon, or the like. It is also possible to use a regulatory region (e. G., A region of interest) by modification, insertion or deletion of a sequence using a locational method (e. G., Homologous recombination) or a random event based method (e. G. Transposon mutagenesis or REMI) For example, a promoter, a repressor, and / or an enhancer may be modified to adjust the activity of a gene of interest. In addition, a nucleic acid sequence which functions as a transcription terminator may be inserted into one or more coding regions 3 'of the present invention; Such terminators are known in the art and are described, for example, in Winnacker, E.L. (1987) From Genes to Clones-Introduction to Gene Technology. VCH: Weinheim.
[175] Example 6 Evaluation of Expression of Mutant Proteins
[176] Observation of the activity of the protein in host cells transformed with the mutated protein revealed that the mutant protein is expressed in a similar manner and in a similar manner as the wild-type protein. A useful method for identifying the transcription level of a mutant gene (an indicator of the amount of mRNA available for translation into a gene product) is Northern blotting (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York), which involves extracting the entire RNA of a biological culture and spreading it on a gel, delivering it to a stable matrix, devising a binding to the gene of interest, Radioactive or chemiluminescent tag), so that the binding of the probe and its binding amount also indicate the presence and amount of the mRNA of the gene of interest. This information is evidence of the degree of transcription of the mutant gene. The intracellular total RNA of Corynebacterium glutamicum is described, for example, in Bormann, E.R. et al. (1992) Mol. Microbiol. 6: 317-326. ≪ / RTI >
[177] To assess the presence or the relative amount of translated protein from this mRNA, standard techniques such as Western blotting can be used (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley : New York]). In this method, whole proteins in cells are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe such as an antibody that specifically binds to a desired protein. Typically, the probe is tagged with a chemiluminescent or colorimetric label that can be easily detected. The presence and amount of the observed label indicates the presence and amount of the desired mutant protein in the cell.
[178] Example 7: Growth of genetically modified Corynebacterium glutamicum Cultivation medium and culture conditions
[179] Genetically modified Corynebacteria are cultured in synthetic or natural growth media. Numerous different growth media of coryneform bacteria are known in the art and are also readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32: 205-210), von der Osten The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et. (1998) al., eds., Springer-Verlag). The medium consists of one or more carbon sources, a nitrogen source, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars such as monosaccharides, disaccharides, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. In addition, the sugar may be supplied to the medium through a complex compound such as molasses or other by-products produced during sugar purification. It may also be advantageous to supply a mixture of different carbon sources. Other possible sources of carbon are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid. The nitrogen source is typically an organic or inorganic nitrogen compound, or a material containing such a compound. Examples of the nitrogen source include ammonia gas or ammonia salts such as NH ₄ Cl or (NH ₄ ) ₂ SO ₄ and NH ₄ OH, nitrates, urea, amino acids or steep liquor, soy flour, soy protein, yeast extract, And a complex nitrogen source such as meat extract.
[180] Examples of inorganic salt compounds that can be contained in the medium include hydrochloride, phosphate, or sulfate of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. The chelate compound can be added to the medium to keep the metal ions in solution. Particularly useful chelate compounds include dihydroxyphenols such as catechol or protocatechuate, or organic acids such as citric acid. In addition, the medium usually contains other growth factors such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often arise from complex media components such as yeast extract, molasses, corn oil, and the like. The exact composition of the media compound varies greatly according to subsequent experiments and is determined separately for each specific case. Information on optimization of media can be found in [Applied Microbiol. Physiology, A Practical Approach (eds. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It may also be selected from growth media from the manufacturer, such as Standard 1 (Merck product) or BHI (Grain Leaf, DIFCO product).
[181] All media components are sterilized by heat treatment (1.5 bar and 121 ° C for 20 minutes) or sterile filtration. These ingredients can be sterilized at once, or sterilized separately if necessary. All media components are present from the beginning of the culture, or may be added continuously or batchwise, as the case may be.
[182] Culture conditions are defined separately for each experiment. The temperature should be between 15 ° C and 45 ° C. The temperature can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably about 7.0, and the medium may be added with buffer to maintain it. An example of a buffer that can be used for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES, etc. may be used alternatively or simultaneously. In addition, during culturing by the addition of NaOH or NH ₄ OH it is also possible to maintain a constant culture pH of water. When complex media components such as yeast extract are used, many complex compounds can reduce the need for additional buffers because of their high buffering capacity. When a fermenter is used for culturing microorganisms, the pH may be controlled using ammonia gas.
[183] The incubation time range is typically several hours to several days. This time is chosen to maximize the amount of product accumulated in the broth. The disclosed culture experiments can be performed in various containers such as microtiter plates of different sizes, glass tubes, glass flasks or glass or metal fermenters. To screen a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks with or without baffles. The desired growth medium is preferably filled in a 100 ml shake flask at 10% (volume ratio). The flasks should be shaken (amplitude 25 mm) on a rotary shaker at a speed range of 100-300 rpm. The loss due to evaporation can be reduced by keeping the atmosphere moist; Alternatively, the amount of loss due to evaporation must be accurately corrected.
[184] When testing genetically modified clones, a control clone containing an unmodified control clone or a basic plasmid without any insert should also be tested. 5 g / L yeast extract, 5 g / L meat extract, 22 g / L urea, 10 g / L polypeptone, 5 g / L yeast extract, 10 g / L glucose, 2.5 g / L NaCl, L agar, adjusted to pH 6.8 with 2 M NaOH) is inoculated into the medium to an OD _{600 of} 0.5-1.5. The medium inoculation is carried out by introducing a saline suspension of Corynebacterium glutamicum cells from a CM plate or by adding a pre-culture of the bacteria.
[185] Example 8: In vitro analysis of the function of mutant proteins
[186] Methods of measuring enzymatic activity and kinetic parameters are well established in the art. Experiments to determine the activity of any given modified enzyme should be tailored to the specific activity of the wild-type enzyme (a person skilled in the art can perform this well). Examples of enzymatic structures, reaction rates, principles, methods and applications, as well as specific examples of enzymatic activity and methods of measuring the activity of many enzymes, can be found in the following references: Dixon, M. et al. , and Webb, EC, (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism. Freeman: New York], Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: SanFrancisco], Price, NC, Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford, Boyer, PD, ed. (1983) The Enzymes, 3 ^rd ed. Academic Press: New York], Bisswanger, H., (1994) Enzymkinetik, 2 ^nd ed. VCH: Weinheim (ISBN 3527300325), Bergmeyer, HU, Bergmeyer, J., Grassl, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3 ^rd ed., Vol. I-XII, Verlag Chemie: Weinheim, and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes. VCH: Weinheim, p. 352-363].
[187] Protein activity binding to DNA can be measured using several well established methods such as DNA band-displacement analysis (sometimes referred to as gel retardation assays). The effect of these proteins on the expression of other molecules can be assessed using a reporter gene assay (see, for example, Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 and the references cited in this document And the like). Reporter gene testing systems using enzymes such as -galactosidase, green fluorescent proteins, and the like are well known and well established for application to prokaryotic and eukaryotic cells.
[188] Measurement of the activity of membrane transport proteins is described in Gennis, R.B. (1989) " Pores, Channels and Transporters ", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137, 199-234, and 270-322.
[189] Example 9: Analysis of the effect of mutant proteins on the desired product production
[190] The effect of genetic modification in Corynebacterium glutamicum on the production of a desired compound (e. G., Amino acid) can be determined by culturing the modified microorganism under suitable conditions (e. G., As described above) It is possible to analyze and evaluate the medium component and / or the intracellular component involved in the increase of production of the product (i.e., amino acid). Such analytical techniques are well known to those skilled in the art and include analytical chromatographic methods such as spectroscopic analysis, thin layer chromatography, various staining methods, enzymatic and microbiological methods, and high performance liquid chromatography methods (see, for example, [Ullman, Encyclopedia of Industrial Chemistry, vol. A2, pp. 89-90 and p.443-613, VCH: Weinheim (1985)], Fallon, A. et al., (1987) VCH: Weinheim < / RTI > et al. (1993) John et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons, Kennedy, JF and Cabral, JMS (1992) Recovery processes for biological materials, John Wiley and Sons, [Shaeiwitz, JA and Henry, JD (1988) Biochemical separations, in: Ulmann's Encycl VCH: Weinheim], and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
[191] Not only the measurement of the final fermentation product, but also the analysis of other components in the metabolic pathway used for the production of the desired compound, such as intermediates and byproducts, the measurement of the overall compound yield, yield and / or production efficiency is also possible. Analytical methods include the determination of nutrients (eg, sugars, hydrocarbons, nitrogen sources, phosphates and other ions) in the medium, the composition and growth of biomass, the generation of common metabolites in the biosynthetic pathway, Gas measurements produced during fermentation, and so on. Standard methods for such measurements are described in Applied Microbial Physiology, A Practical Approach, PMRhodes and PF Stanbury, eds., IRL Press, pp. 103-129, 131-163, and 165-192 (ISBN: 0199635773) Reference cited in the literature].
[192] Example 10: Purification of Desired Product from Corynebacterium glutamicum Cultures
[193] Various methods known in the art can be used to recover the desired product from Corynebacterium glutamicum cells or supernatants of the cultures described above. If the desired product is not secreted from the cells, the cells can be harvested by slow centrifugation of the culture and lysed by standard techniques such as mechanical force or ultrasonication. Cell debris is removed by centrifugation and the soluble protein Is left to be used for further purification of the desired compound. When the product is secreted from Corynebacterium glutamicum cells, the culture is centrifuged at low speed to remove the cells and the supernatant is left for further purification.
[194] The upper layer portion by any of the above two purification methods is applied to chromatography using a suitable resin so that the desired molecule remains on the chromatographic resin but many impurities in the sample remain or the impurities remain in the resin, Not to remain. If desired, these chromatographic steps can be repeated using the same or different chromatographic resins. Those skilled in the art will be well versed in selecting the appropriate chromatographic resin and applying it most effectively to the purification of a particular molecule. The purified product can be concentrated by filtration or ultrafiltration to a temperature at which the stability of the product is maximized.
[195] The purification methods known in the art are various and are not limited to the purification methods described above. Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).
[196] Identification and purity of the isolated compound can be evaluated by standard techniques in the art. These include high performance liquid chromatography (HPLC), spectrometry, staining, thin layer chromatography, NIRS, enzymatic analysis, or microbiological methods. Such assay methods are discussed in the following references: Patek et al. (1994) Appl. Environ.Microbiol. 60: 133-140, Malakhova et al. (1996) Biotekhnologiya 11: 27-32, Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70], Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p.89-90, p.521-540, p.540547, p.559-566, p. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons, and Fallon, A. et < RTI ID = 0.0 > al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol.
[197] Example 11: Analysis of gene sequence of the present invention
[198] Sequence comparison and determination of the homology (%) between two sequences is a technique known in the art, which is described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68) by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77). The algorithm is described in Altschul, et al. (1990) J. Mol. Biol. 215: 403-10] (version 2.0). A nucleotide sequence homologous to the PTS nucleic acid molecule of the present invention can be obtained by searching BLAST nucleotides with an NBLAST program of score = 100 and wordlength = 12. The amino acid sequence homologous to the PTS protein molecule of the present invention can be obtained by searching BLAST protein with XBLAST program of score = 50 and wordlength = 3. Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402], a gap alignment for comparison can be obtained using a gap BLAST. Those skilled in the art will know how to optimize the parameters of the program (e.g., XBLAST and NBLAST) for specific sequences to be analyzed when using BLAST and gap BLAST programs.
[199] Other examples of mathematical algorithms used for sequence comparison are described in Meyers and Miller (1988) Comput. Appl. Biosci. 4: 11-17]. This algorithm is introduced in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, the PAM 120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. Additional sequencing algorithms are known in the art, see Torelli and Robotti (1994) Comput. Appl. Biosci. 10: 3-5], and ADVANCE and ADAM., Described by Pearson and Lipman (1988) P.N.A.S. 85: 2444-8.
[200] Also, a comparison of homology (%) between two amino acid sequences was made using a Blosum 62 matrix or a PAM250 matrix and a GCG software package using gap weights of 12, 10, 8, 6, or 4 and a length of 2, 3, (available at http://www.gcg.com) using the GAP program. A comparison of homologies (%) between two nucleic acid sequences can be performed using the GAP program in the GCG software package with standard parameters such as gap weight 50 and length weight 3.
[201] A gene sequence of the present invention and a gene sequence of Genbank were compared and analyzed using techniques known in the art (see, for example, Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. ≪ / RTI > John Wiley and Sons: New York). The gene sequence of the present invention and the genbank sequence of GenBank were compared in a three-step method. In the first step, each of the sequences of the present invention was subjected to BLASTN analysis (e.g., topical alignment analysis) for the nucleotide sequence of GenBank, and the top 500 hits were left for further analysis. A FASTA search was then performed on the 500 hits (e.g., combined local and global alignment analysis to align the defined regions of the sequence). Then, using the GAP program in the GCG software package (using standard parameters), each gene sequence of the invention was overall sorted into each of the top three FASTA hits. In order to obtain accurate results, the lengths of the sequences selected in GenBank were adjusted to the length of the corresponding sequences using methods known in the art. The results of the analysis are shown in Table 4. The data thus obtained was identical to the data obtained by comparing each gene of the present invention with each reference sequence of GenBank by performing only GAP (general) analysis alone, but when compared with the database-wide GAP (general) Usage time was much lower. Among the sequences of the present invention, those that are not aligned are represented by cutoff values without alignment information in Table 4. < tb > < TABLE > It will also be understood by those skilled in the art that the GAP alignment homology (%) in Table 4 is listed in the European numeral format under the heading "homology (%) (GAP)", where ',' means decimal point. For example, in this stage, the value of "40,345" means "40.345%".
[202] Example 12 Preparation and Execution of DNA Microarray
[203] The sequences of the present invention are well known in the art and are described in Schena, M. et al. (1995) Science 270: 467 (DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48], and DeRisi < RTI ID = 0.0 > , JL et al. (1997) Science 278: 680-686), and the like.
[204] DNA microarrays are solid or flexible supports composed of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules can be attached to this surface in a sequential manner. After appropriately labeled, other nucleic acids or nucleic acid mixtures can be hybridized to the fixed nucleic acid molecule, and the label can be used to monitor and measure the individual signal strength of the hybridized molecules in the defined region. With this method, it is possible to simultaneously quantify the amount or absolute amount of all nucleic acids or the selected nucleic acid in the applied nucleic acid sample or mixture. Therefore, DNA microarrays enable simultaneous analysis of large amounts (over 6,800) of nucleic acid expression (see, for example, Schena, M. (1996) BioEssays 18 (5): 427-431).
[205] The sequence of the present invention can be used to design an oligonucleotide primer capable of amplifying a limited region of one or more Corynebacterium glutamicum genes by a nucleic acid amplification reaction such as a polymerase chain reaction. PCR products were prepared as described above (also see, for example, Schena, M. et al. (1995) Science 270: 467-470) by selection and design of 5 'or 3' oligonucleotide primers or appropriate linkers, Lt; / RTI > can also be covalently attached to the surface of the support medium.
[206] In addition, nucleic acid microarrays are described in Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367. ≪ / RTI > According to the photolithographic method, precisely defined regions of the matrix are exposed to light. This activates a light-labile protecting group that causes nucleotide addition, but does not cause any deformation in the light-shielded region. Different oligonucleotides are synthesized at positions defined by subsequent protection and photoactivating cycles. Small restriction regions of the gene of the present invention can be synthesized by solid state oligonucleotide synthesis on a microarray.
[207] The nucleic acid molecule of the present invention present in a sample or a mixture of nucleotides may hybridize with the microarray. These nucleic acid molecules can be labeled according to standard methods. Briefly, isotopically or fluorescently labeled nucleotides are incorporated, for example, during reverse transcription or during DNA synthesis to label nucleic acid molecules (e.g., mRNA or DNA molecules). Methods for hybridizing labeled nucleic acids to microarrays can be found in the literature (e.g. Schena, M. et al. (1995) supra), Wodicka, L. et al. (1997) And DeSaizieu A. et al. (1998), supra). The method of detection and quantification of the hybridized molecules depends on the specific label incorporated. Radioactive labels are described, for example, in Schena, M. et al. (1995) supra. Fluorescent labels can be detected, for example, by the method described in Shalon et al. (1996) Genome Research 6: 639-645.
[208] As described above, the sequence of the present invention can be applied to the DNA microarray technology to compare and analyze Corynebacterium glutamicum or other Corynebacteria of different strains. For example, mutant studies between strains based on individual transcript profiles and identification of genes important for specific and / or desired strain characteristics such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid sequencing. In addition, nucleic acid sequencing techniques can be used to compare gene expression profiles of the invention during the fermentation reaction process.
[209] Example 13: Kinetic analysis of a protein population in a cell (proteomics)
[210] The genes, compositions and methods of the present invention can be applied to the study of protein interactions and kinetics, referred to as " proteomics ". The population of proteins of interest includes the entire protein population of Corynebacterium glutamicum (e.g., as compared to a population of other organisms), certain environmental or metabolic conditions (e. G., During fermentation, at high or low temperatures, Or a high or low pH), or a population of proteins that are active during a particular period of growth and development, and the like.
[211] Gel electrophoresis, and the like. Intracellular proteins can be obtained by lysis or extraction, and can be separated from each other using various electrophoresis techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins primarily based on their molecular weights. Isoelectric focusing Polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins according to their isoelectric point (meaning not only the amino acid sequence but also the post-translational modifications of the protein). In addition, more preferred protein assays are sequential sequencing of both IEF-PAGE and SDS-PAGE, known as 2D-gel electrophoresis (see for example Hermann et al. (1998) Electrophoresis 19: 3217-3221) , Electrophoresis 19: 1193-1202, Langen et al. (1997) Electrophoresis 18: 1184-1192, Antelmann et al. (1997) Electrophoresis 18: 1451-1462 Lt; / RTI > Proteins may also be isolated using other separation techniques known in the art, such as capillary gel electrophoresis.
[212] In this way, isolated proteins can be visualized using standard techniques such as staining or labeling. Suitable dyes are known in the art and include fluorescent dyes such as Coomassie Brilliant Blue, Silver Dye, or Sypro Ruby (Molecular Probes). Radioactive example a labeled amino acids or other protein precursors (for example, ³⁵ S- methionine, ³⁵ S- cysteine and ¹⁴ amino acids, ¹⁵ amino acid N-, ¹⁵ NO ₃ or ¹⁵ NH ₄ ⁺ or a ¹³ C- labeled C- labeled Amino acid) is included in the Corynebacterium glutamicum culture medium, they can be labeled before the proteins are separated from the cells. Similarly, fluorescent labels can be used. Such labeled proteins can be extracted, isolated and isolated according to the techniques described above.
[213] The protein visualized by the above technique can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be measured quantitatively using an optical method or the like and compared to the amount of other proteins in the same gel or other gel. The gel-like proteins can be compared through optical comparisons, spectroscopy, image scanning and gel analysis, or the use of photographic films and screens. These techniques are well known in the art.
[214] To determine what an arbitrary given protein is, one can use direct sequence analysis or other standard techniques. (E.g., MALDI or ESI techniques (e. G., Langen et al. (1997)), as well as other methods, such as N- and / or C- terminal amino acid sequencing Electrophoresis 18: 1184-1192). Using these techniques, the protein sequences provided herein can be used to identify Corynebacterium glutamicum proteins.
[215] The information obtained from the above method can be used to determine the presence, activity or modification of the protein between different samples from various biological conditions (e.g., different organisms, time of fermentation, media conditions, or biotope between each other) Can be compared. The data obtained by the experiment alone or in combination with other techniques can be used to compare reactions of various organisms in a given state (e.g., metabolic state), increase productivity of a microorganism producing microorganism, or increase production efficiency of fine chemicals It can be applied for various purposes.
[216] Equivalent method
[217] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, as well as many experimental methods equivalent to the specific embodiments of the invention described herein. These equivalent methods will be included in the claims below.

权利要求:
Claims (38)
[1" claim-type="Currently amended] Except for the nucleic acid molecule composed of the F-labeled gene shown in Table 1, except for the nucleotide sequence of the isolated nucleic acid encoding phosphoenolpyruvate: sugar phosphotransferase system protein from Corynebacterium glutamicum Molecule, or part thereof.
[2" claim-type="Currently amended] The method of claim 1, wherein the phosphoenolpyruvate: sugar phosphotransferase system protein is glucose, sucrose, mannose, fructose, mannitol, raffinose, ribulose, ribose, lactose, maltose, sorbose, Wherein the protein is a protein selected from the group consisting of proteins involved in the transport of sorbitol, xylose, and galactose.
[3" claim-type="Currently amended] An isolated Corynebacterium glutamicum nucleic acid molecule, or a part thereof, selected from the group consisting of the sequences listed in the odd numbers of the Sequence Listing, except for the nucleic acid molecule consisting of the F-labeled gene listed in Table 1.
[4" claim-type="Currently amended] An isolated nucleic acid molecule encoding a polypeptide sequence selected from the group consisting of sequences listed in even numbers of the sequence listing, except for the nucleic acid molecule comprising the F-labeled gene set forth in Table 1.
[5" claim-type="Currently amended] An isolated nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide selected from the group of amino acid sequences consisting of sequences listed in even numbers of the sequence listing, except for the nucleic acid molecule comprising the F-labeled gene set forth in Table 1.
[6" claim-type="Currently amended] An isolated nucleic acid molecule comprising a nucleotide sequence having at least 50% homology with a nucleotide sequence selected from the group consisting of the sequences described in the odd-numbered sequence of the sequence listing, except for the nucleic acid molecule consisting of the F- Or part thereof.
[7" claim-type="Currently amended] An isolated nucleic acid comprising a fragment of at least 15 nucleotides of a nucleic acid comprising a nucleotide sequence selected from the group consisting of the sequences described in the odd numbers of the Sequence Listing except for the nucleic acid molecule consisting of the F- molecule.
[8" claim-type="Currently amended] 8. An isolated nucleic acid molecule which hybridizes under stringent conditions to the nucleic acid molecule of any one of claims 1 to 7.
[9" claim-type="Currently amended] 9. An isolated nucleic acid molecule comprising a nucleic acid molecule according to any one of claims 1 to 8 or a part thereof, and a nucleotide sequence encoding a heterologous polypeptide.
[10" claim-type="Currently amended] 10. A vector comprising the nucleic acid molecule of any one of claims 1 to 9.
[11" claim-type="Currently amended] 11. The vector according to claim 10, which is an expression vector.
[12" claim-type="Currently amended] 12. A host cell transfected with the expression vector of claim 11.
[13" claim-type="Currently amended] 13. The host cell according to claim 12, wherein the cell is a microorganism.
[14" claim-type="Currently amended] 14. The host cell of claim 13, wherein the cell is in the genus Corynebacterium or Brevibacterium .
[15" claim-type="Currently amended] 13. The host cell of claim 12, wherein the expression of the nucleic acid molecule controls the production of fine chemicals from the cell.
[16" claim-type="Currently amended] 16. The method of claim 15, wherein the fine chemical is selected from the group consisting of organic acids, proteinogenic amino acids, non-proteinogenic amino acids, purine bases, pyrimidine bases, nucleosides, nucleotides, lipids, saturated fatty acids, unsaturated fatty acids, diols, carbohydrates, A compound, a vitamin, a cofactor, a polyketide, and an enzyme.
[17" claim-type="Currently amended] A method of producing a polypeptide, comprising culturing the host cell of claim 12 in a suitable culture medium to produce the polypeptide.
[18" claim-type="Currently amended] Isolated phosphoenolpyruvate: a sugar phosphotransferase system polypeptide from Corynebacterium glutamicum, or a part thereof.
[19" claim-type="Currently amended] 19. The method of claim 18, wherein the phosphoenolpyruvate: sugar phosphotransferase system protein is selected from the group consisting of glucose, sucrose, mannose, fructose, mannitol, raffinose, ribulose, ribose, lactose, maltose, A protein involved in the transport of galactose.
[20" claim-type="Currently amended] An isolated polypeptide comprising an amino acid sequence selected from the group consisting of the sequences listed in even numbers of the sequence listing except for the amino acid sequence encoded by the F-labeled gene set forth in Table 1.
[21" claim-type="Currently amended] An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of sequences listed in even numbers of the sequence listing except for the amino acid sequence encoded by the F- , Or parts thereof.
[22" claim-type="Currently amended] 22. The isolated polypeptide according to any one of claims 18 to 21, further comprising a heterologous amino acid sequence.
[23" claim-type="Currently amended] A nucleic acid molecule comprising a nucleotide sequence having at least 50% homology with a nucleic acid selected from the group consisting of the sequences described in the odd-numbered sequences of the sequence listing, except for the nucleic acid molecule consisting of the F- Lt; / RTI > polypeptide.
[24" claim-type="Currently amended] An isolated polypeptide comprising an amino acid sequence that is at least 50% homologous to an amino acid sequence selected from the group consisting of sequences listed in even numbered positions in the sequence listing, except for the amino acid sequence encoded by the F-labeled gene set forth in Table 1.
[25" claim-type="Currently amended] 12. A method of producing a fine chemical, comprising culturing the cell containing the vector of claim 12 to produce a precise chemical.
[26" claim-type="Currently amended] 26. The method of claim 25, wherein the method further comprises recovering fine chemicals from the culture.
[27" claim-type="Currently amended] 26. The method of claim 25, wherein the method further comprises transfecting the cell with the vector of claim 11 to obtain a cell containing the vector.
[28" claim-type="Currently amended] 26. The method of claim 25, wherein the cell is comprised in the genus Corynebacterium or Brevibacterium.
[29" claim-type="Currently amended] 26. The method of claim 25, wherein the cell is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium hercululus, Corynebacterium lilium, Corynebacterium acetoacetylpilum, Corynebacterium aceto glutamicum, Corynebacterium acanthopyrum, Corynebacterium ammoniagenes, Corynebacterium phujocens, Corynebacterium nitrilophylls, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibactum But are not limited to, Terium divaricam, Brevibacterium flavum, Brevibacterium heali, Brevibacterium ketoglutamycum, Brevibacterium ketoserečthum, Brevibacterium lactofermentum, Brevibacterium linen S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae, S. cerevisiae,
[30" claim-type="Currently amended] 26. The method of claim 25, wherein the production of said fine chemical is modulated by expression of nucleic acid molecules from said vector.
[31" claim-type="Currently amended] 26. The method of claim 25, wherein the fine chemical is selected from the group consisting of organic acids, proteinogenic amino acids, non-proteinogenic amino acids, purine bases, pyrimidine bases, nucleosides, nucleotides, lipids, saturated fatty acids, unsaturated fatty acids, diols, carbohydrates, Compounds, vitamins, cofactors, polyketides and enzymes.
[32" claim-type="Currently amended] 26. The method of claim 25, wherein the fine chemical is an amino acid.
[33" claim-type="Currently amended] 33. The method of claim 32 wherein said amino acid is selected from the group consisting of lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine and tryptophan ≪ / RTI >
[34" claim-type="Currently amended] 9. A method of producing a fine chemical, comprising culturing cells in which the genomic DNA has been altered by including the nucleic acid molecule of any one of claims 1 to 9.
[35" claim-type="Currently amended] Detecting the presence or activity of Corynebacterium diphtheria in a subject by detecting in the subject whether one or more of SEQ ID NOS: 1 to 34 of the Sequence Listing is present, except for the F-labeled gene or the sequence encoded thereby, A method for diagnosing the presence or activity of Corynebacterium diphtheria in a subject.
[36" claim-type="Currently amended] A nucleic acid molecule selected from the group consisting of nucleic acid molecules described in odd numbers in the sequence listing, wherein said nucleic acid molecule is disrupted.
[37" claim-type="Currently amended] A host cell comprising a nucleic acid molecule selected from the group consisting of nucleic acid molecules described in odd numbers in the sequence listing including at least one modified nucleic acid from the sequence set forth in the odd numbers of the sequence listing.
[38" claim-type="Currently amended] A nucleic acid molecule selected from the group consisting of nucleic acid molecules described in odd numbers of the sequence listing, wherein the regulatory region of the nucleic acid molecule is modified to a wild-type regulatory region.

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HU0203191A3|2009-01-28|

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JP2007236393A|2007-09-20|

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HU0203191A2|2002-12-28|

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TR200700068T2|2007-03-21|

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EP1246922B1|2010-10-13|

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WO2001002583A3|2001-07-26|

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CN1371420A|2002-09-25|

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JP2007267744A|2007-10-18|

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JP2003512024A|2003-04-02|

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PL359501A1|2004-08-23|

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AU5701400A|2001-01-22|

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KR20060121993A|2006-11-29|

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TR200700067T2|2007-03-21|

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BR0012038A|2002-07-02|

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TR200700069T2|2007-02-21|

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WO2001002583A2|2001-01-11|

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EP2272953A1|2011-01-12|

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AU783697B2|2005-11-24|

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SK18892001A3|2002-09-10|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-07-01|Priority to US14269199P

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1999-07-01|Priority to US60/142,691

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1999-08-23|Priority to US15031099P

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1999-08-23|Priority to US60/150,310

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1999-09-03|Priority to DE19942095.5

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1999-09-03|Priority to DE19942097.1

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1999-09-03|Priority to DE19942095

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1999-09-03|Priority to DE19942097

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2000-06-27|Application filed by 스타르크, 카르크, 바스프 악티엔게젤샤프트

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2000-06-27|Priority to PCT/IB2000/000973

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2002-04-03|Publication of KR20020025099A

优先权:

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申请号 | 申请日 | 专利标题

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US14269199P| true| 1999-07-01|1999-07-01|

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US60/142,691|1999-07-01|

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US15031099P| true| 1999-08-23|1999-08-23|

---

US60/150,310|1999-08-23|

---

DE19942097.1|1999-09-03|

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DE19942095.5|1999-09-03|

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DE19942095|1999-09-03|

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DE19942097|1999-09-03|

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PCT/IB2000/000973|WO2001002583A2|1999-07-01|2000-06-27|Orynebacterium glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins|