AU2007202317A1 - Corynebacterium glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins - Google Patents

Description

P001 Section 29 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Corynebacterium Glutamicum genes encoding phosphoenolpyruvate: sugar phosphotransferase system proteins The following statement is a full description of this invention, including the best method of performing it known to us: CORYNEBACTERIUM GLUTAMICUM GENES ENCODING PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM

PROTEINS

Related Applications This application claims priority to U.S. Provisional Patent Application No.: 60/142,691, filed on July 1, 1999, and also to U.S. Provisional Patent Application No.: 60/150,310, filed on August 23, 1999, incorporated herein in their entirety by this reference. This application also claims priority to German Patent Application No.: 19942095.5, filed on September 3, 1999, and also to German Patent Application No.: 19942097.1, filed on September 3, 1999, incorporated herein in their entirety by this reference.

Background of the Invention Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed 'fine chemicals', include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their production is most conveniently performed through large-scale culture of bacteria developed to produce and secrete large quantities of a particular desired molecule. One particularly useful organism for this purpose is Corynebacterium glutamicum, a gram positive, nonpathogenic bacterium. Through strain selection, a number of mutant strains have been developed which produce an array of desirable compounds. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.

Summary of the Invention The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in C.

1 -2glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as phosphoenolpyruvate:sugar phosphotransferase system (PTS) proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The PTS nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, by fermentation processes. Modulation of the expression of the PTS nucleic acids of the invention, or modification of the sequence of the PTS nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).

The PTS nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glulamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C.

glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.

The PTS nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms.

Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.

The PTS proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, transporting high-energy carbon-containing molecules such as glucose into C. glutamicum, or of participating in intracellular signal transduction in

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this microorganism. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Patent No.

4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species lactofermentum) (Yoshihama et al, J Bacteriol. 162: 591- 597 (1985); Katsumata et al.,J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J.

Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals.

The PTS molecules of the invention may be modified such that the yield, production, and/or efficiency of production of one or more fine chemicals is improved.

For example, by modifying a PTS protein involved in the uptake of glucose such that it is optimized in activity, the quantity of glucose uptake or the rate at which glucose is translocated into the cell may be increased. The breakdown of glucose and other sugars within the cell provides energy that may be used to drive energetically unfavorable biochemical reactions, such as those involved in the biosynthesis of fine chemicals.

This breakdown also provides intermediate and precursor molecules 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 invention, one may therefore increase both the energy available to perform metabolic pathways necessary for the production of one or more fine chemicals, and also the intracellular pools ofmetabolites necessary for such production.

Further, the PTS molecules of the invention may be involved in one or more intracellular signal transduction pathways which may affect the yields and/or rate of production of one or more fine chemical from C glutamicum. For example, proteins necessary for the import of one or more sugars from the extracellular medium HPr, Enzyme I, or a member of an Enzyme II complex) are frequently posttranslationally modified upon the presence of a sufficient quantity of the sugar in the cell, such that they are no longer able to import that sugar. While this quantity of sugar at which the transport system is shut off may be sufficient to sustain the normal functioning of the cell, it may be limiting for the overproduction of the desired fine chemical. Thus, it may be desirable to modify the PTS proteins of the invention such that they are no longer responsive to such negative regulation, thereby permitting greater intracellular concentrations of one or more sugars to be achieved, and, by extension, more efficient production or greater yields of one or more fine chemicals from organisms containing such mutant PTS proteins.

This invention provides novel nucleic acid molecules which encode proteins, referred to herein as phosphoenolpyruvate:sugar phosphotransferase system (PTS) proteins, which are capable of, for example, participating in the import of high-energy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and/or of participating in one or more C. glutamicum intracellular signal transduction pathways. Nucleic acid molecules encoding a PTS protein are referred to herein as PTS nucleic acid molecules. In a preferred embodiment, the PTS protein participates in the import of high-energy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and also may participate in one or more C. glutamicum intracellular signal transduction pathways. Examples of such proteins include those encoded by the genes set forth in Table 1.

The following embodiments, the subject of the invention of this application, are specifically disclosed herein: An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7, or a complement thereof.

An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a complement thereof.

An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a complement thereof.

An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:7, or a complement thereof.

An isolated nucleic acid molecule comprising a fragment of at least contiguous nucleotides of the nucleotide sequence of SEQ ID NO:7, or a complement thereof.

An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:8, or a complement thereof.

An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:8.

An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:7.

An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:8.

An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:8.

An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7.

A host cell comprising the nucleic acid molecule of SEQ ID NO:7, wherein the nucleic acid molecule is disrupted.

A host cell comprising the nucleic acid molecule of SEQ ID NO:7, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:7.

0 A host cell comprising the nucleic acid molecule of SEQ ID NO:7,

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Nc wherein the regulatory region of the nucleic acid molecule is modified Srelative to the wild-type regulatory region of the molecule.

Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding a PTS protein or biologically active portions thereof, as well as nucleic r acid fragments suitable as primers or hybridization probes for the detection or Samplification of PTS-encoding nucleic acid DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth in as the odd-numbered SEQ ID NOs in the Sequence Listing SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set forth, as an odd-numbered SEQ ID NO in the Sequence Listing SEQ ID NO:I, SEQ ID NO:3, SEQ ID NO:5, SEQ ID or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth in as an even-numbered SEQ ID NO in the Sequence Listing SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID The preferred PTS proteins of the present invention also preferably possess at least one of the PTS activities described herein.

In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention a sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains a PTS activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to participate in the import of high-energy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and/or to participate in one or more C. glutamicum intracellular signal transduction pathways. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about or 90% and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an amino acid sequence of the invention an entire amino acid sequence selected from those having an even-numbered SEQ ID NO in the Sequence Listing). In another preferred embodiment, the protein is a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention (encoded by an open reading frame shown in the corresponding odd-numbered SEQ ID NOs in the Sequence Listing SEQ ID NO:1, SEQ ID NO:3, SEQ ID SEQ ID In another preferred embodiment, the isolated nucleic acid molecule is derived from C glutamicum and encodes a protein a PTS fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing) and is able to participate in the import of highenergy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and/or to participate in one or more C. glutamicum intracellular signal transduction pathways, or possesses one or more of the activities set forth in Table 1, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.

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-6- In another embodiment, the isolated nucleic acid molecule is at least nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention a sequence of an oddnumbered SEQ ID NO in the Sequence Listing). Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum PTS protein, or a biologically active portion thereof.

Another aspect of the invention pertains to vectors, recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce a PTS protein by culturing the host cell in a suitable medium. The PTS protein can be then isolated from the medium or the host cell.

Yet another aspect of the invention pertains to a genetically altered microorganism in which a PTS gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by the introduction of a nucleic acid molecule of the invention encoding wild-type or mutated PTS sequence as a transgene. In another embodiment, an endogenous PTS gene within the genome of the microorganism has been altered, functionally disrupted, by homologous recombination with an altered PTS gene. In another embodiment, an endogenous or introduced PTS gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional PTS protein. In still another embodiment, one or more of the regulatory regions a promoter, repressor, or inducer) of a PTS gene in a microorganism has been altered by deletion, truncation, inversion, or point mutation) such that the expression of the PTS gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebaclerium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine being particularly preferred.

In another aspect, the invention provides a method of identifying the presence or activity of Cornyebacterium diphiheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention the sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 34)) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.

Still another aspect of the invention pertains to an isolated PTS protein or a portion, a biologically active portion, thereof. In a preferred embodiment, the isolated PTS protein or portion thereof can participate in the import of high-energy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and also may participate in one or more C. glutamicum intracellular signal transduction pathways. In another preferred embodiment, the isolated PTS protein or portion thereof is sufficiently homologous to an amino acid sequence of the invention a sequence of an evennumbered SEQ ID NO: in the Sequence Listing) such that the protein or portion thereof maintains the ability to participate in the import of high-energy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and /or to participate in one or more C. glutamicum intracellular signal transduction pathways.

The invention also provides an isolated preparation of a PTS protein. In preferred embodiments, the PTS protein comprises an amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).

In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) (encoded by an open reading frame set in a corresponding odd-numbered SEQ ID NO: of the Sequence Listing). In yet another embodiment, the protein is at least about preferably at least about 60%, and more preferably at least about 70%, 80%, or and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an entire amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing). In other embodiments, the isolated PTS protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and is able to participate in the import of highenergy carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and/or to participate in one or more C. glutamicum intracellular signal transduction pathways, or has one or more of the activities set forth in Table 1.

Alternatively, the isolated PTS protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%, 97%, or 99% or more homologous, to a nucleotide sequence of one of the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred that the preferred forms of PTS proteins also have one or more of the PTS bioactivities described herein.

The PTS polypeptide, or a biologically active portion thereof, can be operatively linked to a non-PTS polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the PTS protein alone. In other preferred embodiments, this fusion protein results in increased yields, production, and/or efficiency of production of a desired fine chemical from C glutamicum. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates the production of a desired compound from the cell.

In another aspect, the invention provides methods for screening molecules which modulate the activity ofa PTS protein, either by interacting with the protein itself or a substrate or binding partner of the PTS protein, or by modulating the transcription or translation ofa PTS nucleic acid molecule of the invention.

Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of a PTS nucleic acid molecule of the invention, such that a fine chemical is produced. In a preferred embodiment, this method further includes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expression of a PTS nucleic acid. In another preferred embodiment, this method further includes the step of recovering the fine chemical from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3.

Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates PTS protein activity or PTS nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for the uptake of one or more sugars, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates PTS protein activity can be an cK1 agent which stimulates PTS protein activity or PTS nucleic acid expression. Examples of agents which stimulate PTS protein activity or PTS nucleic acid expression include small molecules, active PTS proteins, and nucleic acids encoding PTS proteins that have Mf: been introduced into the cell. Examples of agents which inhibit PTS activity or O expression include small molecules, and antisense PTS nucleic acid molecules.

Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant PTS gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid is L-lysine.

Detailed Description of the Invention The present invention provides PTS nucleic acid and protein molecules which are involved in the uptake of high-energy carbon molecules sucrose, fructose, or glucose) into C. glutamicum, and may also participate in intracellular signal transduction pathways in this microorganism. The molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms. Such modulation may be due to increased intracellular levels of high-energy molecules needed to produce, ATP, GTP and other molecules utilized to drive energetically unfavorable biochemical reactions in the cell, such as the biosynthesis of a fine chemical. This modulation of fine chemical production may also be due to the fact that the breakdown products of many sugars serve as intermediates or precursors for other biosynthetic pathways, including those of certain fine chemicals. Further, PTS proteins are known to participate in certain intracellular signal transduction pathways which may have regulatory activity for one or more fine chemical metabolic pathways; by manipulating these PTS proteins, one may thereby activate a fine chemical biosynthetic pathways or repress a fine chemical degradation pathway. Aspects of the invention are further explicated below.

I. Fine Chemicals The term 'fine chemical' is art-recognized and includes molecules produced by an organism which have applications in various 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, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm el al., eds. VCH: Weinheim, and references contained therein), lipids, both saturated and unsaturated fatty acids arachidonic acid), diols propane diol, and butane diol), carbohydrates hyaluronic acid and trehalose), aromatic compounds aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, Niki, E. Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.

A. Amino Acid Metabolism and Uses Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms. The term "amino acid" is artrecognized. The proteinogenic amino acids, of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the -11nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids may be in the D- or L- optical configuration, though Lamino acids are generally the only type found in naturally-occurring proteins.

Biosynthetic and degradative pathways of each of the 20 proteinogenic amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3 rd edition, pages 578-590 (1988)). The 'essential' amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), so named because they are generally a nutritional requirement due to the complexity of their biosyntheses, are readily converted by simple biosynthetic pathways to the remaining 11 'nonessential' amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine). Higher animals do retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for normal protein synthesis to occur.

Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetics, agriculture, and pharmaceutical industries. Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine. Glutamate is most commonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine. Glycine, Lmethionine and tryptophan are all utilized in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/ Lmethionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as Nacetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH: Weinheim, 1985.

1 -12- The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for review of bacterial amino acid biosynthesis and regulation thereof, see Umbarger, H.E.(1978) Ann. Rev.

Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of aketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine are each subsequently produced from glutamate. The biosynthesis of serine is a threestep process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by the transferal of the side-chain P-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase.

Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differ only at the final two steps after synthesis of prephenate.

Tryptophan is also produced from these two initial molecules, but its synthesis is an 11 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 final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate. Isoleucine is formed from threonine. A complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.

Amino acids in excess of the protein synthesis needs of the cell cannot be stored, and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L. Biochemistry 3 rd ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in terms of energy, precursor molecules, and the enzymes necessary to synthesize them.

Thus it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3 rd ed. Ch. 24: "Biosynthesis of Amino Acids and Heme" p.

13- 575-600 (1988)). Thus, the output of any particular amino acid is limited by the amount of that amino acid present in the cell.

B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses Vitamins, cofactors, and nutraceuticals comprise another group of molecules which the higher animals have lost the ability to synthesize and so must ingest, although they are readily synthesized by other organisms, such as bacteria. These molecules are either bioactive substances themselves, or are precursors of biologically active substances which may serve as electron carriers or intermediates in a variety of metabolic pathways. Aside from their nutritive value, these compounds also have significant industrial value as coloring agents, antioxidants, and catalysts or other processing aids. (For an overview of the structure, activity, and industrial applications of these compounds, see, for example, Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is artrecognized, and includes nutrients which are required by an organism for normal functioning, but which that organism cannot synthesize by itself. The group of vitamins may encompass cofactors and nutraceutical compounds. The language "cofactor" includes nonproteinaceous compounds required for a normal enzymatic activity to occur. Such compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic. The term "nutraceutical" includes dietary supplements having health benefits in plants and animals, particularly humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been largely characterized (Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley Sons; Ong, Niki, E. Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research Asia, held Sept.

1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).

-14- Thiamin (vitamin B 1 is produced by the chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin B 2 is synthesized from (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively termed 'vitamin B 6 pyridoxine, pyridoxamine, pyridoxaand the commercially used pyridoxin hydrochloride) are all derivatives of the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic i acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl- 1-oxobutyl)-P-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of p-alanine and pantoic acid. The enzymes responsible for the biosynthesis steps for the conversion to pantoic acid, to Palanine and for the condensation to panthotenic acid are known. The metabolically active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds in enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are the precursors of Coenzyme A. These enzymes not only catalyze the formation of panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton, panthenol (provitamin B 5 pantetheine (and its derivatives) and coenzyme A.

Biotin biosynthesis from the precursor molecule pimeloyl-CoA in microorganisms has been studied in detail and several of the genes involved have been identified. Many of the corresponding proteins have been found to also be involved in Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic acid is derived from octanoic acid, and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the a-ketoglutarate dehydrogenase complex. The folates are a group of substances which are all derivatives of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic acid and 6methylpterin. The biosynthesis of folic acid and its derivatives, starting from the metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and pamino-benzoic acid has been studied in detail in certain microorganisms.

Corrinoids (such as the cobalamines and particularly vitamin B 1 2 and porphyrines belong to a group of chemicals characterized by a tetrapyrole ring system.

The biosynthesis of vitamin B 1 2 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also termed 'niacin'. Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by largescale culture of microorganisms, such as riboflavin, Vitamin B 6 pantothenate, and biotin. Only Vitamin B 12 is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The language "purine" or "pyrimidine" includes the nitrogenous bases which are constituents of nucleic acids, co-enzymes, and nucleotides. The term "nucleotide" includes the basic structural units of nucleic acid molecules, which are comprised of a nitrogenous 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 language "nucleoside" includes molecules which serve as precursors to nucleotides, but which are lacking the phosphoric acid moiety that nucleotides possess. By inhibiting the biosynthesis of these molecules, or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; by inhibiting this activity in a fashion targeted to cancerous cells, the ability of tumor cells to divide and replicate may be inhibited. Additionally, there are nucleotides which do not form nucleic acid molecules, but rather serve as energy stores AMP) or as coenzymes FAD and NAD).

Several publications have described the use of these chemicals for these medical indications, by influencing purine and/or pyrimidine metabolism Christopherson, R.I. and Lyons, S.D. (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 been focused on the development of new drugs which can be used, for example, as immunosuppressants or -16anti-proliferants (Smith, (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 have other utilities: as intermediates in the biosynthesis of several fine chemicals thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p. 561- 612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide metabolism are increasingly serving as targets against which chemicals for crop protection, including fungicides, herbicides and insecticides, are developed.

The metabolism of these compounds in bacteria has been characterized (for reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine nucleotide biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology, vol. 42, Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism has been the subject of intensive research, and is essential to the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe disease, such as gout. Purine nucleotides are synthesized from ribose-5-phosphate, in a series of steps through the intermediate compound phosphate (IMP), resulting in the production of guanosine-5'-monophosphate (GMP) or (AMP), from which the triphosphate forms utilized as nucleotides are readily formed. These compounds are also utilized as energy stores, so their degradation provides energy for many different biochemical processes in the cell.

Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-triphosphate (CTP).

The deoxy- forms of all of these nucleotides are produced in a one step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are able to participate in DNA synthesis.

-17- D. Trehalose Metabolism and Uses Trehalose consists of two glucose molecules, bound in a, a-1,l linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) U.S.

Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech. 16: 460- 467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium, from which it can be collected using methods known in the art.

II. The Phosphoenolpyruvate:Sugar Phosphotransferase System The ability of cells to grow and divide rapidly in culture is to a great degree dependent on the extent to which the cells are able to take up and utilize high energy molecules, such as glucose and other sugars. Different transporter proteins exist to transport different carbon sources into the cell. There are transport proteins for sugars, such as glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, or raffinose, and also transport proteins for starch or cellulose degradation products. Other transport systems serve to import alcohols methanol or ethanol), alkanes, fatty acids and organic acids like acetic acid or lactic acid. In bacteria, sugars may be transported into the cell across the cellular membrane by a variety of mechanisms. Aside from the symport of sugars with protons, one of the most commonly utilized processes for sugar uptake is the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). This system not only catalyzes the translocation (with concomitant phosphorylation) of sugars and hexitols, but it also regulates cellular metabolism in response to the availability of carbohydrates. Such PTS systems are ubiquitous in bacteria but do not occur in archaebacteria or eukaryotes.

Functionally, the PTS system consists of two cytoplasmic proteins, Enzyme I and HPr, and a variable number of sugar-specific integral and peripheral membrane transport complexes (each termed 'Enzyme II' with a sugar-specific subscript, e.g., 'Enzyme II Gl for the Enzyme 11 complex which binds glucose). Enzymes II specific for mono-, di-, or oligosaccharides, like glucose, fructose, mannose, galactose, ribose, -18sorbose, ribulose, lactose, maltose, sucrose, raffinose, and others are known. Enzyme I transfers phosphoryl groups from phosphoenolpyruvate (PEP) to the phosphoryl carrier protein, HPr. HPr then transfers the phosphoryl groups to the different Enzyme II transport complexes. While the amino acid sequences of Enzyme I and HPr are quite similar in all bacteria, the sequences for PTS transporters can be grouped into structurally unrelated families. Further, the number and homology between these genes vary from bacteria to bacteria. The E. coli genome encodes 38 different PTS proteins, 33 of which are subunits belonging to 22 different transporters. The M genitalium genome contains one gene each for Enzyme I and HPr, and only two genes for PTS transporters. The genomes of T. palladium and C. trachomatis contain genes for Enzyme I- and HPr-like proteins but no PTS transporters.

All PTS transporters consist of three functional units, IIA, IIB, and IIC, which occur either as protein subunits in a complex IIAGICICBGlc) or as domains of a single polypeptide chain IICBAGICN^A). IIA and IIB sequentially transfer phosphoryl groups from HPr to the transported sugars. IIC contains the sugar binding site, and spans the inner membrane six or eight times. Sugar translocation is coupled to the transient phosphorylation of the IIB domain. Enzyme I, HPr, and IIA are phosphorylated at histidine residues, while IIB subunits are phosphorylated at either cysteine or histidine residues, depending on the particular transporter involved.

Phosphorylation of the sugar being imported has the advantage of blocking the diffusion of the sugar back through the cellular membrane to the extracellular medium, since the charged phosphate group cannot readily traverse the hydrophobic core of the membrane.

Some PTS proteins play a role in intracellular signal transduction in addition to their function in the active transport of sugars. These subunits regulate their targets either allosterically, or by phosphorylation. Their regulatory activity varies with the degree of their phosphorylation the ratio of the non-phosphorylated to the phosphorylated form), which in turn varies with the ratio of sugar-dependent dephosphorylation and phosphoenolpyruvate-dependent rephosphorylation. Examples of such intracellular regulation by PTS proteins in E. coli include the inhibition of glycerol kinase by dephosphorylated IIAGIC, and the activation of adenylate cyclase by the phosphorylated version of this protein. Also, the HPr and the 1IB domains of some transporters in these microorganisms regulate gene expression by reversible 19phosphorylation of transcription antiterminators. In gram-positive bacteria, the activity of HPr is modulated by HPr-specific serine kinases and phosphatases. For example, HPr phosphorylated at serine-46 functions as a co-repressor of the transcriptional repressor CcpA. Lastly, it has been found that unphosphorylated Enzyme I inhibits the sensor kinase CheA of the bacterial chemotaxis machinery, providing a direct link between the sugar binding and transport systems of the bacterium and those systems governing movement of the bacterium (Sonenshein, A. et al., eds. Bacillus subtilis and other gram-positive bacteria. ASM: Washington, Neidhardt, et al., eds. (1996) Escherichia coli and Salmonella. ASM Press: Washington, Lengeler et al., (1999).

Biology of Prokaryotes. Section II, pp. 68-87, Thieme Verlag: Stuttgart).

111. Elements and Methods of the Invention The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as PTS nucleic acid and protein molecules, which participate in the uptake of high-energy carbon molecules glucose, sucrose, and fructose) into C. glutamicum, and may also participate in one or more intracellular signal transduction pathways in these microorganisms. In one embodiment, the PTS molecules function to import high-energy carbon molecules into the cell, where the energy produced by their degradation may be utilized to power less energetically favorable biochemical reactions, and their degradation products may serve as intermediates and precursors for a number of other metabolic pathways. In another embodiment, the PTS molecules may participate in one or more intracellular signal transduction pathways, wherein the presence of a modified form of a PTS molecule a phosphorylated PTS protein) may participate in a signal transduction cascade which regulates one or more cellular processes. In a preferred embodiment, the activity of the PTS molecules of the present invention has an impact on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the PTS molecules of the invention are modulated in activity, such that the yield, production or efficiency of production of one or more fine chemicals from C. glutamicum is also modulated.

The language, "PTS protein" or "PTS polypeptide" includes proteins which participate in the uptake of one or more high-energy carbon compounds mono-, di, or oligosaccharides, such as fructose, mannose, sucrose, glucose, raffinose, galactose, ribose, lactose, maltose, and ribulose) from the extracellular medium to the interior of the cell. Such PTS proteins may also participate in one or more intracellular signal transduction pathways, such as, but not limited to, those governing the uptake of different sugars into the cell. Examples of PTS proteins include those encoded by the PTS genes set forth in Table 1 and by the odd-numbered SEQ ID NOs. For general references pertaining to the PTS system, see: Stryer, L. (1988) Biochemistry. Chapter 37: "Membrane Transport", W.H. Freeman: New York, p. 959-961; Damrnell, J. et al.

(1990) Molecular Cell Biology Scientific American Books: New York, p. 552-553, and Michal, ed. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Chapter 15 "Special Bacterial Metabolism". The terms "PTS gene" or "PTS nucleic acid sequence" include nucleic acid sequences encoding a PTS protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions. Examples of PTS genes include those set forth in Table I. The terms "production" or "productivity" are art-recognized and include the concentration of the fermentation product (for example, the desired fine chemical) formed within a given time and a given fermentation volume kg product per hour per liter). The term "efficiency of production" includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical). The term "yield" or "product/carbon yield" is art-recognized and includes the efficiency of the conversion of the carbon source into the product fine chemical). This is generally written as, for example, kg product per kg carbon source. By increasing the yield or production of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. The terms "biosynthesis" or a "biosynthetic pathway" are art-recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds in what may be a multistep and highly regulated process. The terms "degradation" or a "degradation pathway" are art-recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process. The language "metabolism" is art-recognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, -21 then, the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound. The language "transport" or "import" is art-recognized and includes the facilitated movement of one or more molecules across a cellular membrane through which the molecule would otherwise be unable to pass.

In another embodiment, the PTS molecules of the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. Using recombinant genetic techniques, one or more of the PTS proteins of the invention may be manipulated such that its function is modulated. For example, a protein involved in the PTS-mediated import of glucose may be altered such that it is optimized in activity, and the PTS system for the importation of glucose may thus be able to translocate increased amounts of glucose into the cell.

Since glucose molecules are utilized not only for energy to drive energetically unfavorable biochemical reactions, such as fine chemical biosyntheses, but also as precursors and intermediates in a number of fine chemical biosynthetic pathways serine is synthesized from 3-phosphoglycerate). In each case, the overall yield or rate of production of one of these desired fine chemicals may be increased, either by increasing the energy available for such production to occur, or by increasing the availability of compounds necessary for such production to take place.

Further, many PTS proteins are known to play key roles in intracellular signal transduction pathways which regulate cellular metabolism and sugar uptake in keeping with the availability of carbon sources. For example, it is known that an increased intracellular level of fructose 1,6-bisphosphate (a compound produced during glycolysis) results in the phosphorylation of a serine residue on HPr which prevents this protein from serving as a phosphoryl donor in any PTS sugar transport process, thereby blocking further sugar uptake. By mutagenizing HPr such that this serine residue cannot be phosphorylated, one may constitutively activate HPr and thereby increase sugar transport into the cell, which in turn will ensure greater intracellular energy stores and intermediate/precursor molecules for the biosynthesis of one or more desired fine chemicals.

The isolated nucleic acid sequences of the invention are contained within the genome of a Corynebaclerium glutamicum strain available through the American Type -22- Culture Collection, given designation ATCC 13032. The nucleotide sequence of the isolated C. glutamicum PTS DNAs and the predicted amino acid sequences of the C.

glutamicum PTS proteins are shown in the Sequence Listing as odd-numbered SEQ ID NOs and even-numbered SEQ ID NOs, respectively.

Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode metabolic pathway proteins.

The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention the sequence of an even-numbered SEQ ID NO of the Sequence Listing). As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.

The PTS protein or a biologically active portion or fragment thereof of the invention can participate in the transport of high-energy carbon-containing molecules such as glucose into C. glutamicum, or can participate in intracellular signal transduction in this microorganism, or may have one or more of the activities set forth in Table 1.

Various aspects of the invention are described in further detail in the following subsections: A. Isolated Nucleic Acid Molecules One aspect of the invention pertains to isolated nucleic acid molecules that encode PTS polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of PTS-encoding nucleic acid PTS DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules cDNA or genomic DNA) and RNA molecules mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 100 nucleotides 0 -23of sequence upstream from the 5' end of the coding region and at least about nucleotides of sequence downstream from the 3'end of the coding region of the gene.

The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic Sacid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank jthe nucleic acid sequences located at the 5' and 3' ends of the nucleic acid) in the O genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated PTS nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived a C. glutamicum cell). Moreover, an "isolated" nucleic acid molecule, such as a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a C. glutamicum PTS DNA can be isolated from a C. glutamicum library using all or portion of one of the odd-numbered SEQ ID NO sequences of the Sequence Listing as a hybridization probe and standard hybridization techniques as described in Sambrook, Fritsh, E. and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence). For example, mRNA can be isolated from normal endothelial cells by the guanidinium- -24thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can be prepared using reverse transcriptase Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, c available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Sequence Listing. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and Sappropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a PTS nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the invention, as set forth in the Sequence Listing, correspond to the Corynebacterium glutamicum PTS DNAs of the invention. This DNA comprises sequences encoding PTS proteins the "coding region", indicated in each oddnumbered SEQ ID NO: sequence in the Sequence Listing), as well as 5' untranslated sequences and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID NO: in the Sequence Listing. Alternatively, the nucleic acid molecule can comprise only the coding region of any of the nucleic acid sequences of the Sequence Listing.

For the purposes of this application, it will be understood that each of the nucleic acid and amino acid sequences set forth in the Sequence Listing has an identifying RXA, RXN, RXS, or RXC number having the designation "RXA", "RXN", "RXS", or "RXC" followed by 5 digits RXA01503, RXN01299, RXS00315, or RXC00953). Each of the nucleic acid sequences comprises up to three parts: a 5' upstream region, a coding region, and a downstream region. Each of these three regions is identified by the same RXA, RXN, RXS, or RXC designation to eliminate confusion. The recitation "one of the odd-numbered sequences of the Sequence Listing" then, refers to any of the nucleic acid sequences in the Sequence Listing, which may be also be distinguished by their differing RXA, RXN, RXS, or RXC designations. The coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also set forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence For example, the coding region for RXA02229 is set forth in SEQ ID NO:1, while the amino acid sequence which it encodes is set forth as SEQ ID NO:2. The sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, RXS, or RXC designations as the amino acid molecules which they encode, such that they can be readily correlated. For example, the amino acid sequences designated RXA01503, RXN01299, RXS00315, and RXC00953 are translations of the coding regions of the nucleotide sequence of nucleic acid molecules RXA01503, RXN01299, RXS00315, and RXC00953, respectively. The correspondence between the RXA, RXN, RXS, and RXC nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs, is set forth in Table 1. For example, as set forth in Table 1, the nucleotide sequence of RXN01299 is SEQ ID NO: 7, and the corresponding amino acid sequence is SEQ ID NO:8.

Several of the genes of the invention are "F-designated genes". An F-designated gene includes those genes set forth in Table 1 which have an in front of the RXA, RXN, RXS, or RXC designation. For example, SEQ ID NO:3, designated, as indicated on Table 1, as "F RXA00315", is an F-designated gene, as are SEQ ID NOs: 9, 11, and 13 (designated on Table 1 as "F RXA01299", "F RXA01883", and "F RXA01889", respectively).

In one embodiment, the nucleic acid molecules of the present invention are not intended to include C. glutamicum those compiled in Table 2. In the case of the dapD gene, a sequence for this gene was published in Wehrmann, et al. (1998) J.

Bacteriol. 180(12): 3159-3165. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region.

In another preferred embodiment, an 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 a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences of the invention is one which is sufficiently complementary to one of the nucleotide sequences shown in the Sequence -26- Listing the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid molecule of the c invention comprises a nucleotide sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 0 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence of the invention a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. Ranges and identity values intermediate to the above-recited ranges, 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof.

Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of the sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a PTS protein. The nucleotide sequences determined from the cloning of the PTS genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning PTS homologues in other cell types and organisms, as well as PTS homologues from other Corynebacteria or related species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the nucleotide sequences of the invention a sequence of one of the oddnumbered SEQ ID NOs of the Sequence Listing, an anti-sense sequence of one of these -27sequences or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone PTS homologues.

Probes based on the PTS nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used as a part of a diagnostic test kit for identifying cells which misexpress a PTS protein, such as by measuring a level of a PTS-encoding nucleic acid in a sample of cells detecting PTS mRNA levels or determining whether a genomic PTS gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention a sequence of an evennumbered SEQ ID NO of the Sequence Listing), such that the protein or portion thereof maintains the ability to participate in the transport ofhigh-energy carbon molecules (such as glucose) into C. glutamicum, and may also participate in one or more intracellular signal transduction pathways. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent an amino acid residue which has a similar side chain as an amino acid residue in a sequence of one of the evennumbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid sequence of the invention such that the protein or portion thereof is capable of transporting high-energy carbon-containing molecules such as glucose into C.

glutamicum, and may also participate in intracellular signal transduction in this microorganism. Protein members of such metabolic pathways, as described herein, function to transport high-energy carbon-containing molecules such as glucose into C.

glutamicum, and may also participate in intracellular signal transduction in this microorganism. Examples of such activities are also described herein. Thus, "the function of a PTS protein" contributes to the overall functioning and/or regulation of one or more phosphoenolpyruvate-based sugar transport pathway, and /or contributes, either directly or indirectly, to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of PTS protein activities are set forth in Table 1.

-28- In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).

Portions of proteins encoded by the PTS nucleic acid molecules of the invention are preferably biologically active portions of one of the PTS proteins. As used herein, the term "biologically active portion ofa PTS protein" is intended to include a portion, a domain/motif, of a PTS protein that is capable of transporting high-energy carbon-containing molecules such as glucose into C. glutamicum, or of participating in intracellular signal transduction in this microorganism, or has an activity as set forth in Table 1. To determine whether a PTS protein or a biologically active portion thereof can participate in the transportation of high-energy carbon-containing molecules such as glucose into C. glutamicum, or can participate in intracellular signal transduction in this microorganism, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary skill in the art, as detailed in Example 8 of the Exemplification.

Additional nucleic acid fragments encoding biologically active portions of a PTS protein can be prepared by isolating a portion of one of the amino acid sequences of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing), expressing the encoded portion of the PTS protein or peptide by recombinant expression in vitro) and assessing the activity of the encoded portion of the PTS protein or peptide.

The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences of the invention a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the genetic code and thus encode the same PTS protein as that encoded by the nucleotide sequences of the invention. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the Sequence Listing an even-numbered SEQ ID In a still further embodiment, the nucleic acid molecule of the invention encodes a full length C.

-29glutamicum protein which is substantially homologous to an amino acid sequence of the invention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing).

It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to the present invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art a Genbank sequence (or the protein encoded by such a sequence) set forth in Tables 2 or For example, the invention includes a nucleotide sequence which is greater than and/or at least 44% identical to the nucleotide sequence designated RXA01503 (SEQ ID NO:5), a nucleotide sequence which is greater than and/or at least 41% identical to the nucleotide sequence designated RXA00951 (SEQ ID NO:15), and a nucleotide sequence which is greater than and/or at least 38% identical to the nucleotide sequence designated RXA01300 (SEQ ID NO:21). One of ordinary skill in the art would be able to calculate the lower threshold of percent identity for any given sequence of the invention by examining the GAP-calculated percent identity scores set forth in Table 4 for each of the three top hits for the given sequence, and by subtracting the highest GAP-calculated percent identity from 100 percent. One of ordinary skill in the art will also appreciate that nucleic acid and amino acid sequences having percent identities greater than the lower threshold so calculated at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical) are also encompassed by the invention.

In addition to the C. glutamicum PTS nucleotide sequences set forth in the Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by those of ordinary skill in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of PTS proteins may exist within a population the C glutamicum population). Such genetic polymorphism in the PTS gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a PTS protein, preferably a C. glutamicum PTS protein. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the PTS gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in PTS that are the result of natural variation and that do not alter the functional activity of PTS proteins are intended to be within the scope of the invention.

Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. glutamicum PTS DNA of the invention can be isolated based on their homology to the C. gluluamicum PTS nucleic acid disclosed herein using the C.

glutamicum DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of an odd-numbered SEQ ID NO: of the Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those of ordinary skill in the art and can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley Sons, N.Y.

(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 0 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a 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 having a nucleotide sequence that occurs in nature -31encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C.

glutamicum PTS protein.

In addition to naturally-occurring variants of the PTS sequence that may exist in C, the population, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the invention, thereby leading to changes in the amino acid sequence of the encoded PTS protein, without altering the functional ability of the PTS protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a nucleotide sequence of the invention. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the PTS proteins an even-numbered SEQ ID NO: of the Sequence Listing) without altering the activity of said PTS protein, whereas an "essential" amino acid residue is required for PTS protein activity. Other amino acid residues, however, those that are not conserved or only semi-conserved in the domain having PTS activity) may not be essential for activity and thus are likely to be amenable to alteration without altering PTS activity.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding PTS proteins that contain changes in amino acid residues that are not essential for PTS activity. Such PTS proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the PTS activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of the invention and is capable of transporting high-energy carbon-containing molecules such as glucose into C. glutamicum, or of participating in intracellular signal transduction in this microorganism, or has one or more activities set forth in Table 1. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ IDNOs of the Sequence Listing, more preferably at least about 60-70% homologous to one of these sequences, even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of these sequences, and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the amino acid sequences of the invention.

-32- To determine the percent homology of two amino acid sequences one of the amino acid sequences of the invention and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence one of the amino acid sequences of the invention) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence a mutant form of the amino acid sequence), then the molecules are homologous at that position as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences homology of identical positions/total of positions x 100).

An isolated nucleic acid molecule encoding a PTS protein homologous to a protein sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the invention such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCRmediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains lysine, arginine, histidine), acidic side chains aspartic acid, glutamic acid), uncharged polar side chains glycine, asparagine, glutaminc, serine, threonine, tyrosine, cysteine), nonpolar side chains alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains threonine, valine, isoleucine) and aromatic side chains tyrosine, phenylalaninc, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a PTS protein is preferably replaced with another amino acid residue from the same -33side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a PTS coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a PTS activity described herein to identify mutants that retain PTS activity. Following mutagenesis of one of the nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).

In addition to the nucleic acid molecules encoding PTS proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, complementary to the coding strand of a double-stranded DNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire PTS coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a PTS protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues the entire coding region of SEQ ID NO. 5 (RXA01503) comprises nucleotides 1 to 249). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding PTS. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids also referred to as 5' and 3' untranslated regions).

Given the coding strand sequences encoding PTS disclosed herein the sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of PTS mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of PTS mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PTS mRNA. An antisense oligonucleotide can be, for -34example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-Dgalactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, acid wybutoxosine, pseudouracil, queosine, 2-thiocytosine, methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid 5-methyl-2-thiouracil, 3-(3-amino-3-N-2carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a PTS protein to thereby inhibit expression of the protein, by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an ct-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual p-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids.

Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-omethylribonucleotide (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).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave PTS mRNA transcripts to thereby inhibit translation of PTS mRNA.

A ribozyme having specificity for a PTS-encoding nucleic acid can be designed based upon the nucleotide sequence of a PTS DNA disclosed herein SEQ ID (RXA01503)). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PTS-encoding mRNA. See, Cech et al. U.S.

Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742. Alternatively, PTS mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.

Alternatively, PTS gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a PTS nucleotide sequence a PTS promoter and/or enhancers) to form triple helical structures that prevent -36transcription of a PTS gene in target cells. See generally, Helene, C. (1991)Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y Acad Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

B. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a PTS protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

However, the invention is intended to include such other forms of expression vectors, such as viral vectors replication defective retroviruses, adenoviruses and adenoassociated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence in an in vitro transcription/translation system or in a -37host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements 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 those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells.

Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, lacI q T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SP02, or X PL, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC1, MFa, AC, P-60, CYCI, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by one of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein PTS proteins, mutant forms of PTS proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of PTS proteins in prokaryotic or eukaryotic cells. For example, PTS genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M.A. el al. (1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel, C.A.M.J.J. et al. (1991) "Heterologous gene expression in filamentous fungi" in: More Gene Manipulations in Fungi, J.W. Bennet L.L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C.A.M.J.J. Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefactiens -mediated transformation ofArabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586), or mammalian cells.

-38- Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the PTS protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin.

Recombinant PTS protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSI, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- III113-B1, Xgtl 1, pBdCl, and pET 1Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89 and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

Target gene expression from the pTrc vector relies on host RNA polymerase -39transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 d vector relies on transcription from a T7 gnl0-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS 174(DE3) from a resident X prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation _of other varieties of bacteria, appropriate vectors may be selected. For example, the C iplasmids pJl101, pIJ 36 4 pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds.

(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PTS protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), 2 i, pAG-1, Yep6, Yepl3, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C.A.M.J.J. Punt, P.J.

(1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York (IBSN 0 444 904018).

Alternatively, the PTS proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells Sf 9 cells) include the pAc series (Smith et al.

(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In another embodiment, the PTS proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, Kemper, 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; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.

For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type tissue-specific regulatory elements are used to express the nucleic acid). Tissuespecific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al.

(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters ofT cell receptors (Winoto and -41 Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters the neurofilament promoter; Byme and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to PTS mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.

The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of 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.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

-42- A host cell can be any prokaryotic or eukaryotic cell. For example, a PTS protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to one of ordinary skill in the art. Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and protein molecules of the invention are set forth in Table 3.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid linear DNA or RNA a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector a plasmid, phage, phasmid, phagemid, transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAEdextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a PTS protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection cells that have incorporated the selectable marker gene will survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of a PTS gene into which a deletion, addition or substitution has been introduced to thereby alter, functionally disrupt, the PTS gene.

Preferably, this PTS gene is a Corynebacterium glutamicum PTS gene, but it can be a -43homologue from a related bacterium or even from a mammalian, yeast, or insect source.

In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous PTS gene is functionally disrupted no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous PTS gene is mutated or otherwise altered but still encodes functional protein the upstream regulatory region can be altered to thereby alter the expression of the endogenous PTS protein). In the homologous recombination vector, the altered portion of the PTS gene is flanked at its 5' and 3' ends by additional nucleic acid of the PTS gene to allow for homologous recombination to occur between the exogenous PTS gene carried by the vector and an endogenous PTS gene in a microorganism. The additional flanking PTS nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see Thomas, and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism by electroporation) and cells in which the introduced PTS gene has homologously recombined with the endogenous PTS gene are selected, using art-known techniques.

In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene.

For example, inclusion of a PTS gene on a vector placing it under control of the lac operon permits expression of the PTS gene only in the presence of IPTG. Such regulatory systems are well known in the art.

In another embodiment, an endogenous PTS gene in a host cell is disrupted by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced PTS gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional PTS protein. In still another embodiment, one or more of the regulatory regions a promoter, repressor, or inducer) of a PTS gene in a microorganism has been altered by deletion, truncation, inversion, or point mutation) such that the expression of the PTS gene is modulated. One of ordinary skill in the art will appreciate that host cells containing -44more than one of the described PTS gene and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present invention.

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce express) a PTS protein. Accordingly, the invention further provides methods for producing PTS proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a PTS protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered PTS protein) in a suitable medium until PTS protein is produced. In another embodiment, the method further comprises isolating PTS proteins from the medium or the host cell.

C. Isolated PTS Proteins Another aspect of the invention pertains to isolated PTS proteins, and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of PTS protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of PTS protein having less than about 30% (by dry weight) of non-PTS protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-PTS protein, still more preferably less than about 10% of non-PTS protein, and most preferably less than about 5% non-PTS protein. When the PTS protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of PTS protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of PTS protein having less than about 30% (by dry weight) of chemical precursors or non-PTS chemicals, more preferably less than about 20% chemical precursors or non-PTS chemicals, still more preferably less than about 10% chemical precursors or non-PTS chemicals, and most preferably less than about 5% chemical precursors or non-PTS chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the PTS protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum PTS protein in a microorganism such as C. glutamicum.

An isolated PTS protein or a portion thereof of the invention can participate in the transport of high-energy carbon-containing molecules such as glucose into C.

glutamicum, and may also participate in intracellular signal transduction in this microorganism, or has one or more of the activities set forth in Table 1. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or portion thereof maintains the ability to transport high-energy carbon-containing molecules such as glucose into C. glulamicum, or to participate in intracellular signal transduction in this microorganism. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, a PTS protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing. In yet another preferred embodiment, the PTS protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, hybridizes under stringent conditions, to a nucleotide sequence of the invention a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing). In still another preferred embodiment, the PTS protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, -46- 99% or more homologous to one of the nucleic acid sequences of the invention, or a portion thereof. Ranges and identity values intermediate to the above-recited values, 70-90% identical or 80-95% identical) are also intended to be encompassed by the ~l present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. The preferred PTS proteins of the present invention also preferably possess at least one of M the PTS activities described herein. For example, a preferred PTS protein of the present O invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can participate in the transport of high-energy carbon-containing molecules such as glucose into C. gluamicum, and may also participate in intracellular signal transduction in this microorganism, or which has one or more of the activities set forth in Table 1.

In other embodiments, the PTS protein is substantially homologous to an amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and retains the functional activity of the protein of one of the amino acid sequences of the invention yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the PTS protein is a protein which comprises an amino acid sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention and which has at least one of the PTS activities described herein. Ranges and identity values intermediate to the above-recited values, 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In another embodiment, the invention pertains to a full length C. gluramicum protein which is substantially homologous to an entire amino acid sequence of the invention.

-47- Biologically active portions of a PTS protein include peptides comprising amino acid sequences derived from the amino acid sequence of a PTS protein, an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to a PTS protein, which include fewer amino acids than a full length PTS protein or the full length protein which is homologous to a PTS protein, and exhibit at least one activity of a PTS protein. Typically, biologically active portions (peptides, peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of a PTS protein. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of a PTS protein include one or more selected domains/motifs or portions thereof having biological activity.

PTS proteins are preferably produced by recombinant DNA techniques. 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 expressed in the host cell. The PTS protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a PTS protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native PTS protein can be isolated from cells endothelial cells), for example using an anti-PTS antibody, which can be produced by standard techniques utilizing a PTS protein or fragment thereof of this invention.

The invention also provides PTS chimeric or fusion proteins. As used herein, a PTS "chimeric protein" or "fusion protein" comprises a PTS polypeptide operatively linked to a non-PTS polypeptide. An "PTS polypeptide" refers to a polypeptide having an amino acid sequence corresponding to PTS, whereas a "non-PTS polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the PTS protein, a protein which is different from the PTS protein and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the PTS polypeptide and the non-PTS polypeptide are fused in-frame to each other. The non- -48- PTS polypeptide can 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 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant PTS proteins. In another embodiment, the fusion protein is a PTS protein containing a heterologous signal sequence at its N-terminus. In certain host cells mammalian host cells), expression and/or secretion of a PTS protein can be increased through use of a heterologous signal sequence.

Preferably, a PTS chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.

Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety a GST polypeptide). A PTSencoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PTS protein.

Homologues of the PTS protein can be generated by mutagenesis, discrete point mutation or truncation of the PTS protein. As used herein, the term "homologue" refers to a variant form of the PTS protein which acts as an agonist or antagonist of the activity of the PTS protein. An agonist of the PTS protein can retain substantially the same, or a subset, of the biological activities of the PTS protein. An antagonist of the PTS protein can inhibit one or more of the activities of the naturally occurring form of the PTS protein, by, for example, competitively binding to a downstream or upstream member of the PTS system which includes the PTS protein. Thus, the C. glutamicum 0 -49- PTS protein and homologues thereof of the present invention may modulate the activity of one or more sugar transport pathways or intracellular signal transduction pathways in Swhich PTS proteins play a role in this microorganism.

In an alternative embodiment, homologues of the PTS protein can be identified by screening combinatorial libraries of mutants, truncation mutants, of the PTS protein for PTS protein agonist or antagonist activity. In one embodiment, a variegated M library of PTS variants is generated by combinatorial mutagenesis at the nucleic acid O level and is encoded by a variegated gene library. A variegated library of PTS variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PTS sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins for phage display) containing the set of PTS sequences therein.

There are a variety of methods which can be used to produce libraries of potential PTS homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential PTS sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the PTS protein coding can be used to generate a variegated population of PTS fragments for screening and subsequent selection of homologues of a PTS protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PTS coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the PTS protein.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of PTS homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify PTS homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In another embodiment, cell based assays can be exploited to analyze a variegated PTS library, using methods well known in the art.

D. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms; mapping of genomes of organisms related to C. glutamicum; identification and localization of C.

glutamicum sequences of interest; evolutionary studies; determination of PTS protein regions required for function; modulation of a PTS protein activity; modulation of the activity of a PTS pathway; and modulation of cellular production of a desired compound, such as a fine chemical.

The PTS nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof. Also, they may be used to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms -51 under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present.

Although Corynebacterium glutamicum itself is nonpathogenic, it is related to pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology. In this disease, a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells; the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease.

Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.

In one embodiment, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium diphlheriae in the subject. C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid and protein molecules in C. glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject.

The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C glutamicum DNA-binding protein binds, the C. glutamicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the -52localization of the fragment to the genome map of C. glutamicum, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.

The PTS nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The sugar uptake system in which the molecules of the invention participate are utilized by a wide variety of bacteria; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.

Manipulation of the PTS nucleic acid molecules of the invention may result in the production of PTS proteins having functional differences from the wild-type PTS proteins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.

The invention provides methods for screening molecules which modulate the activity of a PTS protein, either by interacting with the protein itself or 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. In such methods, a microorganism expressing one or more PTS proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the PTS protein is assessed.

The PTS molecules of the invention may be modified such that the yield, production, and/or efficiency of production of one or more fine chemicals is improved.

For example, by modifying a PTS protein involved in the uptake of glucose such that it is optimized in activity, the quantity of glucose uptake or the rate at which glucose is translocated into the cell may be increased. The breakdown of glucose and other sugars -53 within the cell provides energy that may be used to drive energetically unfavorable biochemical reactions, such as those involved in the biosynthesis of fine chemicals.

This breakdown also provides intermediate and precursor molecules 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 invention, one may therefore increase both the energy available to perform metabolic pathways necessary for the production of one or more fine chemicals, and also the intracellular pools of metabolites necessary for such production. Conversely, by decreasing the importation of a sugar whose breakdown products include a compound which is used solely in metabolic pathways which compete with pathways utilized for the production of a desired fine chemical for enzymes, cofactors, or intermediates, one may downregulate this pathway and thus perhaps increase production through the desired biosynthetic pathway.

Further, the PTS molecules of the invention may be involved in one or more intracellular signal transduction pathways which may affect the yields and/or rate of production of one or more fine chemical from C. glutamicum. For example, proteins necessary for the import of one or more sugars from the extracellular medium HPr, Enzyme I, or a member of an Enzyme II complex) are frequently posttranslationally modified upon the presence of a sufficient quantity of the sugar in the cell, such that they are no longer able to import that sugar. An example of this occurs in E. coli, where high intracellular levels of fructose 1,6-bisphosphate result in the phosphorylation of HPr at serine-46, upon which this molecule is no longer able to participate in the transport of any sugar. While this intracellular level of sugar at which the transport system is shut off may be sufficient to sustain the normal functioning of the cell, it may be limiting for the overproduction of the desired fine chemical. Thus, it may be desirable to modify the PTS proteins of the invention such that they are no longer responsive to such negative regulation, thereby permitting greater intracellular concentrations of one or more sugars to be achieved, and, by extension, more efficient production or greater yields of one or more fine chemicals from organisms containing such mutant PTS proteins.

This aforementioned list of mutagenesis strategies for PTS proteins to result in increased yields of a desired compound is not meant to be limiting; variations on these -54mutagenesis strategies will be readily apparent to one of ordinary skill in the art. By these mechanisms, the nucleic acid and protein molecules of the invention may be utilized to generate C. glutamicum or related strains of bacteria expressing mutated PTS nucleic acid and protein molecules such that the yield, production, and/or efficiency of production of a desired compound is improved. This desired compound may be any natural product of C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C. glutamicum strain of the invention.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patent applications, patents, published patent applications, Tables, and the Sequence Listing cited throughout this application are hereby incorporated by reference.

2007202317 22 May 2007 TABLE 1: Genes Included in the Invention PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM Nucleotide Amino Acid Identiication Conti INT Start INT Stop Function SEQ ID NO SEQIN Code 1 2 RXS00315 PTS SYSTEM, SUCROSE-SPECIFIC IIABC COMPONENT (EIIABC-SCR) (SUCROSE- PERMEASE IIABC COMPONENT(PH-OSPHOTRANSFERASE ENZYME 11, ABC COMPONENT) (EC 2.7.1.69) 3 4 F RXAOO315 GROO053 6537 5452 PTS SYSTEM, BETA-GLUCOSIDES-SPECIFIC IIABC COMPONENT (EIIABC-BGL) (BETA- GLUCOSIDES- PERMEASE IIABC COMPONENT) (PHOSPHOTRANSFERASE ENZYME 11. ABC COMPONENT) (EC 2.7.1.69) 6 RXAO 1503 GR00424 10392 10640 PTS SYSTEM, BETA-GLUCOSIDES-SPECIFIC IIABC COMPONENT (EIIABC-BGL) (BETA- GLUCOSIDES- PERMEASE IIABC COMPONENT) (PHOSPHOTRANSFERASE ENZYME 11, ABC COMPONENT) (EC 2.7.1.69) 7 8 RXN01299 W0068 11954 9891 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 9 10 F RXA01299 GR00375 6 4.46 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 11 12 F RXA01883 GR00538 2154 2633 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 13 14 F RXA0 1889 GROO540 77 631 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 16 RXA00951 GR00261 564 172 PTS SYSTEM, MANNITOL (CRYPTIC) -SPECIFIC hIA COMPONENT (EIIA-(C)MTL) (MANNITOL (CRYPTIC)- PERMEASE ItA COMPONENT) (PI-OSPHOTRANSFERASE ENZYME 11. A COMPONENT) (EC 2.7.1.69)

L

17 18 RXN01244 WV0068 14141 15844 PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE (EC 2.7.3.9) 19 20 F RXA01244 GR00359 4837 3329 PHOSPHOENOLPYRUVATE-PROTEIN PH-OSPH-OTRANSFERASE (EC 2.7.3.9) 21 22 RXAO 1300 GR00375 637 903 PHOSPI-OCARRIER PROTEIN HPR 23 24 RXN03002 WV0236 1437 1844 PTS SYSTEM. MANNITOL (CRYPTIC) -SPECIFIC IA COMPONENT (EIIA-{C)MTL) (MANNITOL (CRYPTIC)-PERMEASE RIA COMPONENT) (PHOSPHOTRANSFERASE ENZYME 11, A COMPONENT) (EC 2.7.1.69) 26 RXC00953 WV0260 1834 1082 Membrane Spanning Protein involved in PTS system 27 28 RXCO3001 Membrane Spanning Protein involved in PTS system 29 30 RXN01943 WV0120 4326 6374 PTS SYSTEM, GLUCOSE-SPECIFIC IIABC COMPONENT (EC 2.7.1.69) 31 32 F RXA02191 GRD0642 3395 4633 PHOSPI-OENOLPYRUVATE SUGAR PHOSPHOTRANSFERASE 33 34 F RXA01943 G R00557 3944 3540 crt gene; phosphotransferase system giucose-specilic enzyme III 2007202317 22 May 2007 TABLE 2 Excluded Genes GenBank"'~ Gene Name Gene Function Reference Accession No.

A09073 ppg Phosphoenol pyruvate carboxylase Bachmann, B. et al "DNA fragment coding for phosphoenolpyruvat corboxylase, recombinant DNA carrying said fragment, strains carrying the recombinant DNA and method for producing L-aminino acids using said strains," Patent: EP 0358940-A 3 03/21/90 A45579, Threonine dehydratase Moeckel, B. et al. "Production of L-isoleucine by means of recombinant A45581, micro-organisms with deregulated threonine dehydratase," Patent: WO A45583, 95 19442-A 5 07/20/95 A45585 A45587 AB003 132 murC; ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and characterization of the ftsZ gene from coryneformn bacteria," Biochem. Biophys. Res. Commun., 236(2):383-388 (1997) ABO 15023 murC; flsQ Wachi, M. et al. "A murC gene from Caryneforni bacteria," Appi. Microbiol Bioiechnol, 5](2):223-228 (1999) AB018530 dtsR Kimura, E. et "Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium lactofermentum," Biosci. Biotechnol Biochem., 60(10):1565-1570 (1996) AB018531 dtsRI; dtsR2 AB020624 murl D-glutamnate racemnase AB023377 tkt transketolase AB024708 gltB; gltD Glutamine 2-oxoglutarate aminotransferase large and small subunits AB025424 acn aconitase AB027714 rep Replication AB027715 rep; aad Replication protein; aminoglycoside ______________adenyltransferase AF005242 argC AF005635 glnA Glutamine synihetase AF030405 hisF cyclase AF030520 argG Argininosuccinate synthetase AF03 1518 argF Ornithine carbamolytransferase AF036932 aroD 3-dehydroquinate dlehydratase AF038548 pyc IPyruvate carboxylaseI 2007202317 22 May 2007 Tale2 cotinued) AF038651I dciAE; apt; ret Dipeptide-binding protein; adenine Wehmeier, L. et at. "The role of the Corynebacterium gltamicum rel gene in phosphor ibosylItrans ferase; GTP (p)ppGpp metabolism," Microbiology, 144:1853-1862 (1998) pyrophosphokinase AF04 1436 argR Arginine repressor AF045998 impA Inositot monophosphate phosphatase AF048764 argHi Argininosuccinate tyase AF049897 argC; argi; argB; N-acetytglutamnytphosphate reductase; argD; argF; argR; ornithine acetyltransferase; NargG; argH acetylglutamnate kinase; acetytomithine transminase; ornithine carbamoyltrasferase; arginine repressor; argininosuccinate synthase; _____________argininosuccinate tyase AF050 109 inhA Enoyt-acyl carr ier protein reductase AF050 166 hisG ATP phosphoribosyltransferase 1846 hisA Phosphoribosylforfnmi o-5-amino- 1phosphoribosyl-4-imidazolecarboxam ide isom erase AF052652 metA Homnoserine 0-acetyltransferase Park, S. et at. "Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in Corynebacterium glutamicum," Mo!.

8(3):286-294 (1998) AF053071 aroB Dehydroquinate synthetase AF060558 hisH Glutamine amidotransferase AF086704 hisE Phosphoribosyt-ATP- _____________pyrophosphohydrotase AFI 14233 aroA 5-enolpyruvylsh ik imate 3-phosphate synthase AFt 16184 panD L-aspartate-al ph a-decarboxytlase precursor Dusch, N. et at. "Expression of the Corynebacterium glutamicum panD gene encoding L-as partate-atIph a-d ecarboxytlase leads to pantothenate overproduction in Escherichia coti," App!. Environ. Microbiol., 65(4)1530- 1539 (1999) AF124518 aroD; aroE 3-dehydroquinase; shikimate dehydrogenase AF124600 aroC; aroK; aroB; Chorismate synthase; shikimate kinase; 3pepQ dehydroquinate synthase; putative -TF-15897 i~hAcytoplasmic peptidase AF145898 inhA 2007202317 22 May 2007 Table 2 (continued) AJQO01436 ectP Transport of ectoine, glycine betaine, Peter, H. et A. "Corynebacterium glutamicum is equipped with four secondary praline carriers for compatible solutes: Identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoinelproline/glycine carrier, EctP," J BacterioL, 180(22):6005-6012 (1998) AJO04934 dapD Tetrahydrodipicolinate succinylase Wehrmann, A. et al. "Different modes of diaminopimelate synthesis and their (incomplete') role in cell wall integrity: A study with Corynebacterium glutamicum," J Bacterial, 180(12):3159-3165 (1998) AJ00732 ppc; secG; amt; ocd; Phosphoenolpyruvate-carboxylase; 7; high soxA affinity amnmonium uptake protein; putative orn ith ine-cyc lodecarboxy lase; sarcosine oxidase AJO 10319 ftsY, glnB, gInD; srp; Involved in cell division;, PH1 protein; Jakoby, M. et al. "Nitrogen regulation in Corynebacteriumn glutamicum; amtP uridylyltransferase (uridylyl-removing Isolation of genes involved in biochemical characterization of corresponding enzmye); signal recognition particle; low proteins," FEM4S MicrohiaL, I173(2):303-3 10 (1999) ammonium uptake protein AJ132968 cat Chloramphenicoi aceteyl transferase AJ224946 mqo L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Corynebacterium Eur. J Biochem., 254(2):395-403 (1998) AJ238250 ndh NADH A;0238703 porA Porin Lichtinger, T. et al. "Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum: The channel is formed by a low molecular mass polypeptide," Biochemistry, 37(43): 15024-15032 (1998) D1 7429 Transposable element tS3 1831 Vertes et al."lsolation arnd characterization of 1S3 183 1, a transposable element from Corynebacterium glutamicum," Mo!. Microbial.. 11 (4):739-746 (1994) D84 102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al. "Molecular cloning of the Corynebacterium glutamicum (Brevibacterium lactofermentumn AJ 12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase," Micrabialagy, 142:3347-3354 (1996) E0 1358 hdh; hk Homoserine dehydrogenase; homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: JP_ kinase 1987232392-A 1 10/12/87 -E01359 Upstream of the start codon of homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: JP kinase gene 1987232392-A 2 10/12/87 E0 1375 Tryptophan operon EQ01376 trpL; trpE Leader peptide; anthranilate synthase Matsui, K. et al. "Tryptophan operon, peptide and protein coded thereby, utilization of tryptophan operon gene expression and production of Patent: JP 1987244382-A 1 10124/87 2007202317 22 May 2007 2 confinued £0 1377 Promoter and operator regions of Matsui, K. et al. "Tryptophan operon, peptide and protein coded thereby, tryptophan operon utilization of tryptophan operon gene expression and production of Patent: JP 1987244382-A 1 10/24/87 E03937 Biotin-synthase Hatakeyama, K. et al. "DNA fragment containing gene capable of coding synthetase and its utilization," Patent: JP 1992278088-A 1 10/02/92 E04040 Diamino pelargonic acid aminotransferase Kohama, K. et al. "Gene coding diamninopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1 E04041 Desthiobiotinsynthetase Kohama, K. et al. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1 11/ 18/92 E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding aspartase and utilization thereof," Patent: P 1993030977-A 1 02109/93 £04376 Isocitric acid lyase Katsumnata, R. et al. "Gene manifestation controlling DNA," Patent: JP 3 03/09/93 E04377 Isocitric acid lyase N-terminal fragment Katsumata, R. et al. "Gene manifestation controlling DNA," Patent: JP 3 03/09/93 E04484 Prephenate dehydratase Sotouchi, N. et al. "Production of L-phenylalanine by fermentation," Patent: JP 2 03/30/93 E05108 Aspartokinase Fugono, N. et al. "Gene DNA coding Aspartokinase and its use," Patent: JP 184366-A 1 07/27/93 112 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. "Gene DNA coding dihydrodipicolinic acid synthetase its use," Patent: JP 1993 18437 1-A 1 07/27/93 £05776 Diaminopimelic acid dehydrogenase Kobayashi, M. et al. "Gene DNA coding Diaminopimelic acid dehydrogenase its use," Patent: JP 1993284970-A 1 11/02/93 £05779 Threonine synthase Kohama, K. et al. "Gene DNA coding threonine synthase and its use," Patent: 1993284972-A 1 11/02/93 £061 10 Prephenate dehydratase Kikuchi, T. et al. "Production of L-phenylalanine by fermentation method," JP 199334488 I-A I 12/27/93 £06111 Mutated Prephenate dehydratase Kikuchi, T. et al. "Production of L-phenylalanine by fermentation method," JP 1993344881-A 1 12/27/93 £0-6146 Acetohydroxy acid synthetase lnui, M. et al. "Gene capable of coding Acetohydroxy acid synthetase and its Patent: JP 1993344893-A I 12/27/93 E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase. gene," patent: JP 1994062866-A 1 £06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A I 2007202317 22 May 2007 2 (continued) E06827 Mutated aspartokinase alpha subunit Sugimoto, M. et al "Mutant aspartokinase gene," patent: JP 1994062866-A 1 E07701 secY H-onno, N. et al. "Gene DNA participating in integration of membraneous to membrane," Patent: JP 1994 169780-A 1 06/21/94 E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from inhibition and its utilization," Patent: JP 199426 1766-A 1 09/20/94 E08178, Feedback in hib ition-rel]eased Aspartokinase Sato, Y. et ai. "Genetic DNA capable of coding Aspartokinase released from E08179, feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08 180, E08181, E08 182 E08232 Acetohydroxy-acid isomeroreductase lnui, M. et al. "Gene DNA coding acetohydroxy acid isomeroreductase," Patent: JP 1994277067-A 1 10/04/94 E08234 secE Asai, Y. et al. "Gene DNA coding for translocation machinery of protein," JP 1994277073-A 1 10/04/94 E08643 FT aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA fragment having promoter function in synthetase promoter region coryneform bacterium," Patent: JP 199503 1476-A 1 02/03/95 E08646 Biotin synthetase .Hatakeyama, K. et al. "DNA fragment having promoter function in coryneform bacterium," Patent: JP 199503 1476-A 1 02/03/95 E08649 Aspartase Kohama, K. et al "DNA fragment having promoter function in coryneforin Patent: J P 199503 1478-A 1 02/03/95 E08900 Dihydrodipicolinate reductase Madori, M. et al. "DNA fragment containing gene coding Dihydrodipicolinate acid reductase and utilization thereof," Patent: JP 1995075578-A 1 03/20/95 E08901 Diarniinopimelic acid decarboxylase Madori, M. et al. "DNA fragment containing gene coding Diaminopimelic acid and utilization thereof," Patent: JP 1995075579-A 1 03/20/95 E212594 Serine hydroxymethyltransferase I-latakeyamna, K. et al. "Production of L-trypophan," Patent: JP 199702839 1-A 102/04/97 E 12760, transposase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: E12759, JP 1997070291 -A 03/18/97 E12758 E12764 Arginyl-tRNA synthetase; diamninopimelic Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: decarboxylase JP 1997070291 -A 03/18/97 E12767 Dihydrodipicolinic acid synthetase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: 1997070291-A 03/18/97 E212770 aspartokinase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291 -A 03/18/97 E212773 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP1997070291 I-A 03/18/97 2007202317 22 May 2007 Table 2 (continued) E13655 Glucose-6-phosphate dehydrogenase l-atakeyaina, K. et al. "GlIucose-6- phosphate dehydrogenase and DNA capable of coding the same," Patent: JP 1997224661 I-A 1 09/02/97 LO 1508 INvA Threonine dehydratase Moeckel, B. et al. "Functional and structural analysis of the threonine dehydratase of Corynebacterium glutamicum," J. Bacterial., 174:8065-8072 L07603 EC 4.2.1.15 3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The cloning and nucleotide sequence of Corynebacterium phosphate synthase glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene," FEMS Microbial. Left., 107:223-230 (1993) L09232 I1v13; ilvN; ilvC Acetohydroxy acid synthase large subunit; Keilhauer, C. et al. "Isoleucine synthesis in Corynebacterium glutamicum: Acetohydroxy acid synthase small subunit; molecular analysis of the ilvB-ilvN-i~vC operon," J Bacteriol., 175(17):5595- Acetohydroxy acid isomeroreductase 5603 (1993) L18874 PtsM Phosphoenolpyruvate sugar Fouet, A et al. "Bacillus subtilis sucrose-specific enzyme 11 of the phosphotransferase phosphotransferase system: expression in Escherichia coli and homology to enzymes 11 from enteric bacteria," PNAS USA, 84(24):8773-8777 (1987); Lee, J. K. et al. "Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme If and analyses of the deduced protein.

sequence," FEMS Microbial. Lett., 1 19(1-2):137-145 (1994) L27 123 aceI3 Malate synthase Lee, H-S. et al. "Molecular characterization of aceB, a gene encoding malate synthase in Corynebacterium glutam icum," J Microbial. Biorechnol., (1994) L27 126 Pyruvate kinase Jetten, M. S. et al. "Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum," Appi. Environ. Microbial, 60(7):2501-2507 L28760 aceA Isocitrate lyase L35906 dtxr Diphtheria toxin repressor Oguiza, J.A. et al. "Molecular cloning, DNA sequence analysis, and characterization of the Corynebacteriumn diphtheriae dtxR from Brevibacterium J Bacterial., 1 77(2):465-467 (1995) M 13774 Prephenate dehydratase Follettie, M.T. et al. "Molecular cloning and nucleotide sequence of the glutamicum pheA gene," J Bacterial., 167:695-702 (1986) M 16175 5S rRNA Park, Y-H. et al. "Phylogenetic analysis of the coryneform bacteria by 56 sequences," J. Bacterial, 169:1801-1806 (1987) M 16663 IrpE Anthranilate synthase, 5' end Sano, K. et al. "Structure and function of the trp operon control regions of Brevibacterium lactofermentrm, a glutamic-acid-producing bacterium," Gene, (1987) M 16664 trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and function of the trp operon control regions of Brevibacterium lactofermentum, a glutamic-acid-producing bacterium," Gene.

191-200 (1987) 2007202317 22 May 2007 Table 2 (continued) M25819 Phosphoenolpyruvate carboxylase O'Regan, M. et al. "Cloning and nucleotide sequence of the Phosphoenolpyrvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032," Gene, 77(2):237-251 (1989) M85106 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes," J. Gen.

Microbiol., 138:1167-1175 (1992) M85107, 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive bacteria with a high DNA G+C content are M85108 characterized by a common insertion within their 23S rRNA genes," J. Gen.

Microbiol., 138:1167-1175 (1992) M89931 aecD; bmQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, 1. et al. "The Corynebacterium glutamicum aecD gene encodes a C-S uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination activity that degrades aminoethylcysteine," J. Bacteriol.. 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product," Arch. Microbiol., 169(4):303-312 (1998) S59299 trp Leader gene (promoter) Herry, D.M. et al. "Cloning of the trp gene cluster from a tryptophanhyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence," Appl Environ. Microbiol., 59(3):791-799 (1993) U 1545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican, L.K. (1994) Complete nucleotide sequence of the Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology Department, University College Galway, Ireland.

U 13922 cgllM; cgllR; clgllR Putative type II 5-cytosoine Schafer, A. et al. "Cloning and characterization ofa DNA region encoding a methyltransferase; putative type II stress-sensitive restriction system from Corynebacterium glutamicum ATCC restriction endonuclease; putative type I or 13032 and analysis of its role in intergeneric conjugation with Escherichia type III restriction endonuclease coli," J. Bacteriol., 176(23):7309-7319 (1994); Schafer, A. et al. "The Corynebacterium glutamicum cgllM gene encoding a 5-cytosine in an McrBCdeficient Escherichia coli strain," Gene, 203(2):95-101 (1997) U14965 recA U31224 ppx Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., 178(15):4412-4419 (1996) U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol, 178(15):4412-4419 (1996) U31230 obg; proB; unkdh ?;gamma glutamyl kinase;similar to D- Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., dehydrogenases 178(15):4412-4419 (1996) 2007202317 22 May 2007 Table 2 (continued) U31281 bioB Biotin synthase Serebriiskii, "Two new members of the bio B superfamily: Cloning, sequencing and expression of bio B genes of Methylobacillus flagellatumn and Corynebacterium glutamicum," Gene, 175:15-22 (1996) UJ35023 thtR; accBC Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A Corynebacterium glutamnicumn gene encoding a two-domain carb~oxylase protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins," Arch. Microbil., 166(2);76-82 (1996) U43535 cmr Multidrug resistance protein Jager, W. et ai. "A Corynebacterium glutamicumn gene conferring multidrug resistance in the heterologous host Escherichia coli," J Bacteriol., 179(7):2449-2451I (1997) U43536 clpB Heat shock ATP-binding protein U53587 aphA-3 3 -aminoglycoside phosphotransferase U89648 Corynebacterium glutamicum unidentified sequence involved in histidine biosynthesis, partial X04960 trpA; trpB; trpC; trpD; Tryptophan operon Matsui, K. et al. "Complete nucleotidle and deduced amino acid sequences of trpE; trpG; trpL the Brevibacterium lactofermentumn tryptophan operon," Nucleic Acids Res., 14(24):101 13-10114 (1986) X07563 lys A DAP decarboxylase (meso-diam inopime late Yeh, P. et al. "Nucleic sequence of the lysA gene of Corynebacterium decarboxylase, EC 4.1.1.20) glutamicum and possible mechanisms for modulation of its expression," Mt.

Genet., 212(1):112-119 (1988) X 14234 EC 4.1.1.31 Phosphoenolpyruvate carboxylase Eikmanns, B.i. et al. "The Phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and expression," Mat. Gen. Genet., 218(2):330-339 (1989); Lepiniec, L. et al.

"Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function molecular evolution," Plant. Mat. Bil., 21 (3):487-502 (1993) Xl17313 fda Fructose-bisphosphate aldolase Von der Osten, C.H. et at. "Molecular cloning, nucleotide sequence and finestructural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutam icum fiructose-I1, 6-biphosphate aldolase to class I and 11 aldolases," Mt. Microbil., X53993 dapA L-2, 3-dihydrodipicotinate synthetase (EC Bonnassie, S. et at. "Nucleic sequence of the dapA gene from Corynebacterium glutam icum," Nucleic Acids Res., 18(21 ):6421I (1990) X54223 AttB-related site Cianciotto, N. et at. "DNA sequence homology between att B3-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum and the attP site of lambdacorynephage," FEMS. Microbial, 66:299-302 (1990) X54740 argS; lysA Arginyl-IRNA synthetase; Diaminopimelate Marcel, T. et at. "Nucleotide sequence and organization of the upstream region decarboxylase of the Corynebacterium glutamnicumn lysA gene," Mat. Micrabiat., 4(l1): 1819- 1830(1990) 2007202317 22 May 2007 2 (continued) X55994 trpL; trpE Putative leader peptide; anthranilate Heery, D.M. et al. "Nucleotide sequence of the Corynebacterium glutamicum component I trpE gene," Nucleic Acids Res., 18(23):7138 (1990) X56037 thrC Threonine synthase Han, K.S. et al. "The molecular structure of the Corynebacterium glutanicum synthase gene," Mol. Microbial., 4(10):1693-1702 (1990) X560t75 attB-related site Attachment site Cianciotto, N. et al. "DNA sequence homology between att S-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum and the attP site of lambdacorynephage," FEMS. Microbial, Leif., 66:299-302 (1990) X57226 lysC-alpha; lysC-beta;, Aspartokinase-alpha subunit; Kalinowski, J. et al. "Genetic and biochemical analysis of the Aspartokinase asd Aspartokinase-beta subunit; aspartate beta from Corynebacterium glutamicum," McI. Microbio., 5(5):1 197-1204 (1991); semialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in ____________Corynebacterium glutamicum," Moi. Gen. Genet., 224(3):317-324 (1990) X59403 gap;pgk; tpi G lyceraidehyde-3 -phosphate; Eikmanns, B.J. "Identification, sequence analysis, and expression of a phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum gene cluster encoding the three glycolytic isomerase enzymes glIyceraldehyde-3 -phosphate dlehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomeras," J. Bacterial., 174(19):6076.6086 (1992) X59404 gdh Glutamate dlehydrogenase Bormann, E.R. et al. "Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamnate dehydrogenase," Mol. Microbial., 6(3):3 17-326 X603 12 lysI L-lysine permease Seep-Feldhaus, A.H. et al. "Molecular analysis of the Corynebacterium glutamicum lysI gene involved in lysine uptake," Mo!. Microbial., 5(12):2995- (1991) X66078 cop I Psi protein Joliff, G. et al. "Cloning and nucleotidle sequence of the csplI gene encoding PSI, one of the two major secreted proteins of Corynebacteriumn glutarnicum: The deduced N-termninal region of PSI is similar to the Mycobacterium antigen complex," Mol. Micrabial., 6(1 6):2349-2362 (1992) X661 12 git Citrate synthase Eikmanns, B.J. et al. "Cloning sequence, expression and transcriptional analysis of the Corynebacteriumn glutamicumn g~tA gene encoding citrate Microbial, 140:1817-1828 (1994) X67737 d apB Dihydrodipicolinate reductase X69 103 csp2 Surface layer protein PS2 Peyret, J.L. et al. "Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum," Mol. Microbial., (1993) X69104 153 related insertion element Bonamy, C. et al. "Identification of IS51206, a Corynebacterium glutamicum 153-related insertion sequence and phylogenetic analysis," Mal. Microbial., (1994) 2007202317 22 May 2007 2 (continued) X70959 leuA Isopropylmalate synthase Patek, M. et al. "Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis," Appl. Environ. Microbial., 60(1): 133-140 (1994) X71489 icd Isocitrate dehydrogenase (NADP4) Eikmanns, BiJ. et al. "Cloning sequence analysis, expression, and inactivation of the Corynebacteriumn glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme," J Bacierial..

(1995) X72855 GDHA Glutamate dehydrogenase (NADP+) X 75083, mtrA 5-methyltryptophan resistance Heery, D.M. et al. "A sequence from a tryptophan-hyperproducing strain of X70594 Corynebacterium glutamicum encoding resistance to Biaphys. Res. Commun., 201(3):1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA mutant strains of Corynebacterium glutamicum and Brevibacteriumn lactofermentum," Appi.

Biolechno., 42(4):575-580 (1994) X7554 acA; hiX artal Iocitatelyas; ?Reinscheid, D.J. et al. "Characterization of the isocitrate lyase gene from X7554 acA; hiXPartal socirat lyae;?Corynebacterium glutamicum and biochemical analysis of the enzyme," J Bacterial., 176(12):3474-3483 (1994) 78 7S5 ATPase beta-subunit Ludwig, W. et al. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes," Antanie Van Leeuwenhaek, 64:285-305 (1993) X77034 tuf Elongation factor Tu Ludwig, W. et al. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factorlTu and ATP-synthase beta-subunit genes," Antonie Van Leeuwenhoec 64:285-305 (1993) Y773 84 recA Billman-Jacobe, H. "Nucleotide sequence of a recA gene from ____________Corynebacterium glutamicum," DNA Seq., 4(6):403-404 (1994) X78491 aceB Malate synthase Reinscheid, D.J. et al. "Malate synthase from Corynebacteriumn glutamicum pta-ack operon encoding phosphotransacetylase: sequence analysis," Microbiology, 140:3099-3 108 (1 994) X80629 16S rDNA 16S ribosomal RNA Rainey, F.A. et al. "Phylogenetic analysis of the genera Rhodococcus and Norcardia and evidence for the evolutionary origin of the genus Norcardia *from within the radiation of Rhodococcus species," Microbial, 14 1:523-528 (1995) X81 191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al. "Structure of the gluABCD cluster encoding the gluD glutamate uptake system of Corynebacteriumn glutamicum," J Bacterial, 177(5):1 152-I1158 (1995) X81379 dapE Succinyldiaminopimelate desuccinylase Wehrmann, A. et al. "Analysis of different DNA fragments of Corynebacterium glutamicum complementing dapE of Escherichia coli," 40:3349-56 (1994) 2007202317 22 May 2007 Table 2 (continued) X82061 1 6S rDNA 1 6S ribosomal RNA Ruimy, R. et al. "Phylogeny of the genus Corynebacterium deduced from analyses of small-subunit ribosomal DNA sequences," Int. J. Syst. Bacieriol..

(1995) X82928 asd; IysC Aspartate-semialdehyde dehydrogenase; Serebrijski, 1. et al. "Multicopy suppression by asd gene and osmotic stressdependent complementation by heterologous proA in proA mutants," J.

1 77(24):7255-7260 (1995) X82929 proA Gamma-glutamyl phosphate reductase Serebrijski, 1. et al. "Multicopy suppression by asd gene and osmotic stressdependent complementation by heterologous proA in proA mutants," J.

1 77(24):7255-7260 (1995) X84257 16S rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of the genus Corynebacterium based 16S rRNA gene sequences," Int. J Syst. Bacteriol., 45(4):724-728 (1995) X85965 aroP; dapE Aromatic amino acid permease; Wehrmann et al. "Functional analysis of sequences adjacent to dapE of C.

glutamicum proline reveals the presence of aroP, which encodes the aromatic acid transporter," J Bacterial., 1 77(20):599 1-5993 (1995) X86 157 argB; argC; argD; Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V. et al. "Genes and enzymes of the acetyl cycle of arginine argF; argi glutamyl-phosphate reductase, biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early acetylornithine aminotransferase;I omnithine steps of the arginine pathway," Microbiology, 142:99-108 (1996) carbamoylItrans ferase; glutamate Nacety [trans ferase X89084 pta; ackA Phosphate acetyl trans ferase; acetate kinase Reinscheid, DiJ. et al. "Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding and acetate kinase," Microbiology, 145:503-5 13 (1999) X89850 att B Attachment site Le Marrec, C. et al. "Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus C70,"J Bacterial..

1996-2004 (l1996) X90356 Promoter fragment F I Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90357 Promoter fragment F2 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90358 Promoter fragment FI10 Patek, M. el al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology 142:1297-1309 (1996) X90359 Promoter fragment F 13 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) 2007202317 22 May 2007 Table 2 (continued) X90360 Promoter fragment F22 Patek, M. et al. "Promoters from Corynebacteriumn glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90361 Promoter fragment F34 Patek, M. et al. "Promoters from Corynebacteriumn glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90362 Promoter fragment F37 Patek, M. et al. "Promoters from C. glutamicum: cloning, molecular analysis search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90363 Promoter fragment F45 Pate Mi. et al. "Promoters from Corynebacteriumn glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology (1996) X5N03 64 Promoter fragment F64 Patek, M. et al. "Promoters from Corynebacteriumn glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90365 Promoter fragment F75 Patek, M. et al. 'Promoters from Corynebacterium glutarnicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142: 1297-1309 (1996) X90366 Promoter fragment PFI10I Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90367 Promoter fragment PFI104 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 1297-1309 (1996) X90368 Promoter fragment PF109 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X93513 amrt Ammonium transport system Siewe, R.M. et al. "Functional and genetic characterization of the (methyl) ammonium uptake carrier of Corynebacterium glutamicum," J Biol. Chem., 271(10):5398-5403 (1996) X93514 betP Glycine betaine transport system Peter, H. et al. "Isolation, characterization, and expression of the Corynebacteriumn glutamnicumn betP gene, encoding the transport system for the compatible solute glycine betaine," J Bacteriol., 178(17):5229-5234 (1996) X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of the dapl3-ORF2dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes involved in L-lysine synthesis," Biotechnol. Lett., 19:1113-1117 (1997) X96471I lysE; lysG Lysine exporter protein; Lysine export Vrljic, M. et al. "A new type of transporter with a new type of cellular regulator protein function: L-Iysine export from Corynebacteriumn glutamicum," Mol.

22(5):815-826 (1996) 2007202317 22 May 2007 Table 2 (continued) X96580 panB; panG; xylB 3-methyl-2-oxobutanoate Sahm, H. et at. "D-pantothenate synthesis in Corynebacterium glutamicumn and hydroxymethyltransferase; pantoate-beta- use of panBG and genes encoding L-valine synthesis for D-pantothenate alanine ligase; xylulokinase overproduction," Appi. Environ. Microbial., 65(5): 1973-1979 (1999) X96962 Insertion sequence IS 1207 and transposase X99289 Elongation factor P Ramos, A. et al. "Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofennentumn (Corynebacterium glutamicum ATCC 13869)," Gene, 198:217-222 (1997) YOO 140 thrB Homoserine kinase Mateos, L.M. et al. "Nucleotide sequence of the homoserine kinase (thrB) gene the Brevibacterium lactofermentum," Nucleic Acids Res.. 15(9):3922 (1987) Y00151 ddh Meso-diaminopimelate D-dehydrogenase Ishino, S. et al. "Nucleotide sequence of the meso-diaminopimelate D- (EC 1.4.1.16) dehydrogenase gene from Corynebacterium glutamicum," Nucleic Acids Res., 15(9):3917 (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L.M. et al. "Nucleotide sequence of the homoserine dehydrogenase (thrA) gene of the Brevibacterium lactofermnentum," Nucleic Acids Res., 15(24):10598 (1987) Y00546 horn; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al. "Nucleotide sequence and fine structural analysis of the kinase Corynebacterium glutamicum hom-thrB operon," Mol. Microbiol., 2(1):63-72 (1988) Y08964 murC; ftsQ/divD; ftsZ UPD-N-acetylmuramate-alanine ligase; 1lonrubia, M.P. et al. "Identification, characterization, and chromosomal division initiation protein or cell division organization of the ftsZ gene from Brevibacterium lactofermentum," McI. Gen.

cell division protein Gene., 259(1):97-104 (1998) Y09 163 putP High affinity proline transport system Peter, H. et al. "Isolation of the putP gene of Corynebacterium glutamicumproline and characterization of a low-affinity uptake system for compatible solutes," Arch. Microbial., 168(2): 143-151 (1997) Y09548 pyc Pyruvate carboxylase Peters- Wend isch, P.G. et al. "Pyruvate carboxylase from Corynebacterium glutarnicum: characterization, expression and inactivation of the pyc gene," Mlicrobiology 144:915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et al. "Analysis of the leuB gene from Corynebacterium glutamicum," Appi. Microbial. Biolechnol., 50(1):42-47 (1998) Y 12472 Attachment site bacteriophage Phi- 16 Moreau, S. et al. "Site-specific integration of corynephage Phi-16: The construction of an integration vector," Mficrobial., 145:539-548 (1999) Y12537 proP Proline/ectoine uptake system protein Peter, H. et al. "Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: Identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP," J Bacteriol., 180(22):6005-6012 (1998) 2007202317 22 May 2007 Table 2 (continued) Y13221 gInA Glutamine synthetase I Jakoby, M. et al "Isolation of Corynebacterium glutamicumn ginA gene encoding glutamine synthetase FEMS Microbial Left., 154(l):81-88 (1997) Y 16642 lpd Dihydrolipoamide dehydrogenase Y18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis of the integration functions of φ304L: An module among corynephages," Virology, 255(l): 150-159 (1999) Z21501 argS; lysA Arginyl-tRNA synthetase; diaminopimelate Oguiza, i.A. et al. "A gene encoding arginyl-tRNA synthetase is located in the decarboxylase (partial) upstream region of the lysA gene in Brevibacterium lactofermentum: Regulation of argS-lysA cluster expression by arginine," J 1 75(22):7356-7362 (1993) Z2 1502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et al. "A cluster of three genes (dapA, orf2, and dapB) of dihydrodlipicolinate reductase Brevibacterium lactofermentumn encodes dihydrodipicolinate reductase, and a third polypeptide of unknown function," J Bacteriol, 175(9):2743-2749 Z29563 thrC Threonine synthase Malumbres, M. et al. "Analysis and expression of the thrC gene of the encoded synthase," App!. Environ. Microbiol, 60(7)2209-22 19 (1994) Z46753 16S rDNA Gene for 16S ribosomal RNA Z49822 sigA SigA sigma factor Oguiza, L.A. et al "Multiple sigma factor genes in Brevibacterium lactofermentum: Characterization of sigA and sigB," J Bacterial. 178(2):550- 553 (1996) Z49823 galE; dtxR Catalytic activity UDP-galactose 4- Oguiza, J.A. et al "The galE gene encoding the UDP-galactose 4-epimerase of epimerase; diphtheria toxin regulatory Brevibacterium lactofermentumn is coupled transcriptionally to the dmdR protein gene," Gene, 177:103-107 (1996) Z49824 orf I; sigB SigB sigma factor Oguiza, l.A. et al "Multiple sigma factor genes in Brevibacteriumn lactofennentum: Characterization of sigA and sigB," J. Bacterial., I 78(2):S50- 553 (1996) Z66534 Transposase Correia, A. et al. "Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentumn ATCC 13869," Gene, 170(1):91-94_(1996) _A sequence for this gene was published in the indicated reference. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region.

70 TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention Brevibacteriuni P -Smz ammoniagenes 21054 Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium animoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacterium am moniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518 Brevibacterium flavum BI 11474 Brevibactenium flavum BI 11472 Brevibacterium flavum 21127 Brevibacterium tlavum 21128 Brevibacterium flavum 21427 Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacterium flavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum BI 1477 Brevibacterium flavum BI 11478 Brevibacterium flavum 21127 Brevibacterium flavum BI 11474 Brevibacterium healii 15527 Brevibactenium ketoglutamicum 21004 Brevibactenium ketoglutamicum 21089 Brevibacteriuxn ketosoreductum 21914 Brevibacterium lactofermentum Brevibacterium lactofermentum 74 Brevibacterium lactofermentum 77 Brevibacterium lactofennentum 21798 Brevibacterium lactofermentum 21799 Brevibacterium lactofermnentum 21800 Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B31 1470 Brevibacterium lactofermentum B 11471 71 Brevibacterium lactofermentrm 21086 Brevibacterium lactofermentumn 21420 Brevibacterium lactofermentumn 21086 Brevibacterium lactofermentumn 31269 Brevibacteriwn linens 9174 Brevibacterium linens 19391 Brevibacteriwn linens 8377 Brevibacterium paraffinolyticum 11160 Brevibacterium Spec. ____717.73 Brevibacteriuxn Spec. 717.73 Brevibacteriuni Spec. 14604 Brevibacterium Spec. 21860 Brevibacterium Spec. 21864 Brevibacterium Spec. 21865 Brevibacterium Spec. 21866 Brevibacterium Spec. 19240 Corynebacterium acetoacidophilumn 21476 Corynebacterium acetoacidophilum 13870 Corynebacterium acetoglutamnicumn 811473 Corynebacterium acetoglutamnicumn B11475 Corynebacterium acetoglutamnicumn 15806 Corynebacterium acetoglutamnicumn 21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilum B3671 Corynebacterium ammoniagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacterium glutamnicumn 14067 Corynebacterium glutwnicum 39137 Corynebacterium glutamnicumn 21254 1__ Corynebacterium glutamnicumn 21255 Corynebacterium glutamnicumn 31830 Corynebacterium glutamnicum 13032 Corynebacteriuni glutamnicumn 14305 Corynebacteriuxn glutainicumn 15455 Corynebacterium glutainicumn 13058 Corynebacterium glutamnicumn 13059 Corynebacterium glutamnicumn 13060 Corynebacterium glutamnicurn 21492 Corynebacterium glutamnicumn 21513 Corynebacterium glutamicum 21526 Corynebacterium glutarnicumn 21543 Corynebacterium glutamnicumn 13287 Corynebacterium glutamnicumn 21851 Corynebacterium glutamicumn 21253 Corynebactenium glutamicum 21514 Corynebacterium glutamnicumn 21516 Corynebacterium glutamnicum 21299 Corynebacterium glutamnicum 21300 72 Corynebacterium glutamicum 39684 Corynebacterium glutamicum 21488 Corynebacterium glutamicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 192231 Corynebacterium glutamicum 13869 Corynebacterium glutamicum 21157 Corynebacterium glutainicum 21158 Corynebacterium glutamicum 21159 CorynebacteriuM glutamicum 21355 Corynebacterium glutamicum 31808 Corynebacterium glutamicum 21674 Corynebacterium glutamicum 21562 Corynebacterium glutamicum 21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum 21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum 21567 Corynebacterium glutamnicum 21568 Corynebacterium glutamicum 21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum 21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum 19051 Corynebacterium glutamicum 19052 Corynebacterium glutamicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutainicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutaniicum 19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum 19185 Corynebacterium glutamicum 13286 Corynebacterium glutamicum 21515 Corynebacterium glutamicum 21527 CorynebacteriuM glutamicum 21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum B8183 Corynebacterium glutamicum _B8 182 Corynebacterium glutamicum B12416 Corynebacterium glutamicum B 12417 Corynebacterium glutamicum Corynebacterium glutamicum BI -73 Corynebacterium glutamicum 21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 I1594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088 Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145 Corynebacterium spec. 21857 Corynebacterium spec. 21862 Corynebacterium spec. 21863 ATCC: American Type Culture Collection, Rockville, MD, USA FERM: Fermentation Research Institute, Chiba, Japan NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor Schimmelcultures, Baar, NL NCTC: National Collection of Type Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.

F

2007202317 22 May 2007 TABLE 4: ALIGNMENT RESULTS Lenath Accession Name of Genbank Hit ID O lenoth Genbank Hi~

(NTI

Source of Genbank HI~ Y6 Date of homology Deposit

(GAP)

rxaGO315 1527 GB_8A1:AB007125 GBIN1:CELC47D2 GB-HTG2:AC006732 rxafll503 372 GBPR3:ACOOSO1 9 GB_GSS12:AQ390040 GBGSS5:A07-4231 rxa0l 299 2187 GBEST38;AW047298 GB_RO:AB004056 GB_RO:AB004056 416 GB-BA1:SCJ21 GB_VI:MCU68299 4078 ABOO71 25 Serratia marcescens sisA gene for surface layer protein, complete Serratla marcescens 40.386 cds, isolate 8000.

17381 U64861 Caenorhabditis etegans cosmid C47D2. Caenorhabditis 159453 AC006732 Caenorhabditis elegans clone Y32G9, SEQUENCING IN Caenorhabditis PROGRESS unordered pieces.

188362 AC005019 Homo sapiens SAC clone GS250A16 from 7p21-p22, complete Homo sapiens 680 AQ390040 RPCI11-157C9.TJ RPCI-11 Homo sapiens genomic clone RPCI- Homo sapiens 11 -157C9,genomic survey sequence.

542 A0784231 HS_3087_BICIOT7C CIT Approved Human Genomic Sperm Homo sapiens Library D Homo sapiens genomic clone Plate=3087 CoI=l19 Row=F, genomic survey sequence.

614 AW047296 Ui-M-BH1-amh-e-03-0-UI.sl NIH_BMAP_M_S2 Mus musculus Mus muscutus cONA clone Ui-M-BH1-amh-e-03-0-UI 3% mRNA sequence.

1581 ABDG4056 Rattus norvegicus mRNA for BarI--class homeodomain Raltus norvegic transcription factor. complete cds.

1581 AB004056 Rattus norvegicus mRNA for Ban-I-class homeodomain Rattus norvegic transcription factor, complete cds.

31717 AL109747 Streptomyces coelicolor cosmid J21. Streptomyces 26-MAR-i1998 28-Jul-96 23-Feb,-99 elegans 36,207 elegans 36,436 39,722 27-Aug-98 43,137 21-MAY-1999 37,643 3-Aug-99 41,475 18B-Sep-9 41,031 2-Sep-98 40,717 2-Sep-98 :us :us ;oelicolor 34,913 230278 A3(2) Mouse cytornegalovirus 1 40.097 5-Aug-99 04-DEC-1 996 U68299 Mouse cytomnegalovirus 1 complete genomic sequence.

rxa01244 18B27 rxa0l300 390 rxa0O953 789 GB VI:U93872 133661 U93872 Kaposi's sarcoma-associated herpesvirus glycoprotein M. DNA Kaposrs sarcomareplication protein. glycoprotein, DNA replication protein. FLICE associated herpesvirus inhibitory protein and v-cyclin genes, complete cdls, and tegument GBBA1:AFAPHBHI 4501 M69036 Alcaligenes eutrophus protein H (phbH) and protein I (phbl) Raistonia eutropha genes, complete cdls.

GB-PR3:HSJ836E13 78055 AL050326 Human DNA sequence from clone 836E13 on chromosome 20 Homo sapiens Contains ESTs, STS and GSSs. complete sequence.

GB-EST24:A1170227 409 A1170227 EST216152 Normalized rat lung, Bento Soares Rattus sp. cONA Rattus sp.

clone RLUCF56 3Tend, mRNA sequence.

PGB_-PR3:HUMDODDA 26764 L39874 Homo sapiens deoxycytidylate deaminase gene, complete cds. Homo sapiens GB-PAT:140899 26764 140899 Sequence 1 from patent US 5622851. Unknown.

GB_-PAT:140900 1317 140900 Sequence 2 from patent US 5622851. Unknown.

IGB,,BA1:SCJ21 31717 AL109747 Streptomyces coelicolor cosmid J21. Streptomyces coelicolo 36,029 9-Jul-97 45,624 26-Apr-93 37,303 23-Nov-99 39,098 20-Jan-99 37,644 11 37,644 13-MAY-1 997 37.644 13-MAY-1997 39,398 5-Aug-99 39,610 10-Feb-99 46,753 29-Sep-97 r GB-BA1 :BLTRP GByPAT:EO1 375 7725 X04960 Brevibacteriumn lactofermentumn tryptophan operon.

7726 E01375 DNA sequence of tryptophan operon.

A3(2) Corynebacterium glutamicurn Corynebacterlumn glutamicumn 2007202317 22 May 2007 rxaOl 943 2172 GBBAI:CORPTSMA 2656 GB-BA1:BRLPTSG 3163 GB-BA2:AF045481 2841 Table 4 (continued) L185874 Corynebacterium glutamicum phosphoenolpyruvate sugar phosphotransterase (ptsM) mRNA, complete cds.

L18875 Brevibacterium lactofermentum phosphoenolpyruvate sugar phosphotransterase (ptsG) gene, complete cds.

AF045481 Corynebacterum ammoniagenes glucose permease (ptsG) gene, complete cds.

Corynebactenumr glutamicumn Brevibacterium lactofermentum Corynebacterium ammoniagenes 100O,000 24-Nov-94 84,963 01-OCT-1993 53,558 29-Jul-98 -76- Exemplification Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum ATCC 13032 A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 0 C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I of the original volume of the culture all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l MgSO, x 7H,0, 10 ml/1 KH2PO, solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/1 M12 concentrate (10 g/l 1 g/l NaCI, 2 g/1 MgSO, x 7H 2 0, 0.2 g/l CaCI,, 0.5 g/1 yeast extract (Difco), 10 ml/1 trace-elements-mix (200 mg/1 FeSO, x HO, 10 mg/I ZnSO, x 7 HO, 3 mg/l MnCI, x 4 HO, 30 mg/l HBO 3 20 mg/1 CoCI, x 6 H 2 0, 1 mg/1 NiCI, x 6 H 2 O, 3 mg/l NaMoO, x 2 H,O, 500 mg/1 complexing agent (EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/1 biotin, 0.2 mg/1 folic acid, mg/I p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/l ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/l pyridoxole hydrochloride, 200 mg/l myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37'C, the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HC1, 1 mM EDTA, pH The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution and 0.5 ml NaCI solution (5 M) are added. After adding of proteinase K to a final concentration of 200 gg/ml, the suspension is incubated for ca.18 h at 37 0 C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroformisoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20 0 C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing pg/ml RNaseA and dialysed at 4°C against 1000 ml TE-buffer for at least 3 hours.

During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added. After a min incubation at -200C, the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer. DNA prepared by this procedure could be used for all purposes, including southern blotting or construction of genomic libraries.

EXAMPLE 2: Construction of genomic libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032 Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see Sambrook, J. et al. (1989) "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley Sons).

Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286. Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, and A.J. Sinskey (1994) J. Microbiol. Biotechnol. 4:256-263).

Example 3: DNA Sequencing and Computational Functional Analysis Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using AB1377 sequencing machines (see Fleischman, R.D. et al.

(1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512). Sequencing primers with the following nucleotide sequences were used: 5'-GGAAACAGTATGACCATG-3' (SEQ ID NO.

or 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO. 36).

Example 4: In vivo Mutagenesis In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain -78the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.D.

(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well known to one of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.

Example 5: DNA Transfer Between Escherichia coli and Corynebacterium glutamicum Several Corynebacterium and Brevibacterium species contain endogenous plasmids (as pHM1519 or pBLl) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley Sons) to which a origin or replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E.

coli and C. glutamicum, and which can be used for several purposes, including gene overexpression (for reference, see Yoshihama, M. et al. (1985) J. Bacteriol. 162:591-597, Martin J.F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. et al. (1991) Gene, 102:93-98).

Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and to introduce such a hybrid vectors into strains of Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described e.g. in Schafer, A et al.

-79- (1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough Murray (1983) J. Mol. Biol. 166:1-19).

Genes may be overexpressed in C glutamicum strains using plasmids which comprise pCGI Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, and A. J.

Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).

Aside from the use of replicative plasmids, gene overexpression can also be achieved by integration into the genome. Genomic integration in C. glutamicum or other Corynebacterium or Brevibacterium species may be accomplished by well-known methods, such as homologous recombination with genomic region(s), restriction endonuclease mediated integration (REMI) (see, DE Patent 19823834), or through the use of transposons. It is also possible to modulate the activity of a gene of interest by modifying the regulatory regions a promoter, a repressor, and/or an enhancer) by sequence modification, insertion, or deletion using site-directed methods (such as homologous recombination) or methods based on random events (such as transposon mutagenesis or REMI). Nucleic acid sequences which function as transcriptional terminators may also be inserted 3' to the coding region of one or more genes of the invention; such terminators are well-known in the art and are described, for example, in Winnacker, E.L. (1987) From Genes to Clones Introduction to Gene Technology. VCH: Weinheim.

Example 6: Assessment of the Expression of the Mutant Protein Observations of the activity of a mutated protein in a transformed host cell rely on the fact that the mutant protein is expressed in a similar fashion and in a similar quantity to that of the wild-type protein. A useful method to ascertain the level of transcription of the mutant gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al.

(1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information is evidence of the degree of transcription of the mutant gene. Total cellular RNA can be prepared from Corynebacterium glutamicum by several methods, all well-known in the art, such as that described in Bormann, E.R. et al.

(1992) Mol. Microbiol. 6: 317-326.

To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell.

Example 7: Growth of Genetically Modified Corynebacterium glutamicum Media and Culture Conditions Genetically modified Corynebacteria are cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-210; von der Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE 4,120,867; Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al., eds.

Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be -81 advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH,C1 or (NI-) 2 SO NH,OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.

Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook "Applied Microbiol. Physiology, A Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.

All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121 C) or by sterile filtration. The components can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise.

Culture conditions are defined separately for each experiment. The temperature should be in a range between 15'C and 45C. The temperature can be kept constant or can be altered during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media.

An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It -82is also possible to maintain a constant culture pH through the addition of NaOH or NHIOH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the microorganisms, the pH can also be controlled using gaseous ammonia.

The incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes.

For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 ml shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100 300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.

If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested. The medium is inoculated to an OD 00 oo of 0.5 1.5 using cells grown on agar plates, such as CM plates (10 g/1 glucose, 2,5 g/l NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/1 NaCI, 2 g/l urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30'C. Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.

Example 8 In vitro Analysis of the Function of Mutant Proteins The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one of ordinary skill in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be 83 found, for example, in the following references: Dixon, and Webb, (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.

Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ.

Press: Oxford; Boyer, ed. (1983) The Enzymes, 3 rd ed. Academic Press: New York; Bisswanger, (1994) Enzymkinetik, 2 nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, Bergmeyer, Gral1, 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.

The activity of proteins which bind to DNA can be measured by several wellestablished methods, such as DNA band-shift assays (also called gel retardation assays).

The effect of such proteins on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBOJ. 14: 3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.

The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234; and 270-322.

Example 9: Analysis of Impact of Mutant Protein on the Production of the Desired Product The effect of the genetic modification in C. glutamicum on production of a desired compound (such as an amino acid) can be assessed by growing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product an amino acid). Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, -84- Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al.

(1993) Biotechnology, vol. 3, Chapter III: "Product recovery and purification", page 469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S.

(1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications.) In addition to the measurement of the final product of fermentation, it is also possible to analyze other components of the metabolic pathways utilized for the production of the desired compound, such as intermediates and side-products, to determine the overall productivity of the organism, yield, and/or efficiency of production of the compound. Analysis methods include measurements of nutrient levels in the medium sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways, and measurement of gasses produced during fermentation. Standard methods for these measurements are outlined in Applied Microbial Physiology, A Practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and references cited therein.

Example 10: Purification of the Desired Product from C. glutamicum Culture Recovery of the desired product from the C. glutamicum cells or supematant of the above-described culture can be performed by various methods well known in the art.

If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The cellular debris is removed by centrifugation, and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C. glutamicum cells, then the cells are removed from the culture by low-speed centrifugation, and the supernate fraction is retained for further purification.

The supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One of ordinary skill in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.

There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey, J.E. Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27- 32; and 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. 540- 547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al.

(1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

Example 11: Analysis of the Gene Sequences of the Invention The comparison of sequences and determination of percent homology between two sequences are art-known techniques, and can be accomplished using a mathematical algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad Sci.

USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA -86- 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score 100, wordlength 12 to obtain nucleotide sequences homologous to PTS nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score 50, wordlength 3 to obtain amino acid sequences homologous to PTS protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program XBLAST and NBLAST) for the specific sequence being analyzed.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci. 4: 11- 17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, and include ADVANCE and ADAM. described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.

The percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. The percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parameters, such as a gap weight of 50 and a length weight of 3.

A comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the invention -87were compared to genes present in Genbank in a three-step process. In a first step, a BLASTN analysis a local alignment analysis) was performed for each of the sequences of the invention against the nucleotide sequences present in Genbank, and the top 500 hits were retained for further analysis. A subsequent FASTA search a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits. Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP program in the GCG software package (using standard parameters). In order to obtain correct results, the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4. The resulting data is identical to that which would have been obtained had a GAP (global) analysis alone been performed on each of the genes of the invention in comparison with each of the references in Genbank, but required significantly reduced computational time as compared to such a database-wide GAP (global) analysis. Sequences of the invention for which no alignments above the cutoff values were obtained are indicated on Table 4 by the absence of alignment information.

It will further be understood by one of ordinary skill in the art that the GAP alignment homology percentages set forth in Table 4 under the heading homology (GAP)" are listed in the European numerical format, wherein a represents a decimal point. For example, a value of "40,345" in this column represents "40.345%".

Example 12: Construction and Operation of DNA Microarrays The sequences of the invention may additionally be used in the construction and application of DNA microarrays (the design, methodology, and uses of DNA arrays are well known in the art, and are described, for example, in Schena, M. et al. (1995) Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367; DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L. et al.

(1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label -88may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).

The sequences of the invention may be used to design oligonucleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270: 467-470).

Nucleic acid microarrays may also be constructed by in situ oligonucleotide synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359- 1367. By photolithographic methods, precisely defined regions of the matrix are exposed to light. Protective groups which are photolabile are thereby activated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions. Small, defined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis.

The nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays. These nucleic acid molecules can be labeled according to standard methods. In brief, nucleic acid molecules mRNA molecules or DNA molecules) are labeled by the incorporation of isotopically or fluorescently labeled nucleotides, during reverse transcription or DNA synthesis.

Hybridization of labeled nucleic acids to microarrays is described in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. (1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for example, as -89described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).

The application of the sequences of the invention to DNA microarray technology, as described above, permits comparative analyses of different strains of C.

glutamicum or other Corynebacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the invention during the course of a fermentation reaction are possible using nucleic acid array technology.

Example 13: Analysis of the Dynamics of Cellular Protein Populations (Proteomics) The genes, compositions, and methods of the invention may be applied to study the interactions and dynamics of populations of proteins, termed 'proteomics'. Protein populations of interest include, but are not limited to, the total protein population of C glutamicum in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.

Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety ofelectrophoretic techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largely on the basis of their molecular weight. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein). Another, more preferred method of protein analysis is the consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al.

(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art.

Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors 3SS-methionine, 3SS-cysteine, 4 C-labelled amino acids, 'N-amino acids, 15NO 3 or 5 NHI-4 or 1 C-labelled amino acids) in the medium of C. glutamicum permits the labeling of proteins from these cells prior to their separation. Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.

Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be determined quantitatively using, for example, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and. screens. Such techniques are well-known in the art.

To determine the identity of any given protein, direct sequencing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degradation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, Langen el al. (1997) Electrophoresis 18: 1184-1192)). The protein sequences provided herein can be used for the identification of C glutamicum proteins by these techniques.

The information obtained by these methods can be used to compare patterns of protein presence, activity, or modification between different samples from various biological conditions different organisms, time points of fermentation, media conditions, or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals.

EQUIVALENTS

Those of ordinary skill in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Comprises/comprising and grammatical variations thereof when used in Cc this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

Claims (30)

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7, or a complement thereof.

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a complement thereof.

3. An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a complement thereof.

4. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:7, or a complement thereof. An isolated nucleic acid molecule comprising a fragment of at least contiguous nucleotides of the nucleotide sequence of SEQ ID NO:7, or a complement thereof.

6. An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:8, or a complement thereof.

7. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1-6 and a nucleotide sequence encoding a heterologous polypeptide.

8. A vector comprising the nucleic acid molecule of any one of claims 1-7.

9. The vector of claim 8, which is an expression vector. A host cell transfected with the expression vector of claim 9.

11. The host cell of claim 10, wherein said cell is a microorganism.

12. The host cell of claim 11, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.

13. The host cell of claim 10, wherein the expression of said nucleic acid molecule results in the modulation in production of a fine chemical from said cell.

14. The host cell of claim 13, wherein said fine chemical is selected from the group consisting of: organic acids, proteinogenic and nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides, and enzymes.

15. A method of producing a polypeptide, the method comprising culturing the host cell of claim 10 in an appropriate culture medium to, thereby, produce the polypeptide.

16. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:8.

17. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

18. An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:7.

19. An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:8. An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:8.

21. An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7.

22. The isolated polypeptide of any one of claims 16-21, further comprising a heterologous amino acid sequence.

23. A method for producing a fine chemical, comprising culturing the cell of claim 10 such that the fine chemical is produced.

24. The method of claim 23, wherein said method further comprises the step of recovering the fine chemical from said culture.

25. The method of claim 23, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.

26. The method of claim 23, wherein said cell is selected from the group consisting of: Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacterium nitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum, and those strains of Table 3.

27. The method of claim 23, wherein expression of the nucleic acid molecule from said vector results in modulation of production of said fine chemical.

28. The method of claim 23, wherein said fine chemical is selected from the group consisting of: organic acids, proteinogenic and nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides and enzymes.

29. The method of claim 23, wherein said fine chemical is an amino acid. The method of claim 29, 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.

31. A method for producing a fine chemical, the method comprising culturing a cell whose genomic DNA has been altered by the introduction of a nucleic acid molecule of any one of claims 1-6.

32. A method for diagnosing the presence or activity of Corynebacterium diphtheria, the method comprising detecting the presence of at least one of the nucleic acid molecules of any one of claims 1-6 or the polypeptide molecules of any one of claims 16-21, thereby diagnosing the presence or activity of Corynebacterium diphtheriae.

33. A host cell comprising the nucleic acid molecule of SEQ ID NO:7, wherein the nucleic acid molecule is disrupted.

34. A host cell comprising the nucleic acid molecule of SEQ ID NO:7, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:7. A host cell comprising the nucleic acid molecule of SEQ ID NO:7, wherein the regulatory region of the nucleic acid molecule is modified relative to the wild- type regulatory region of the molecule. BASF AKTIENGESELLSCHAFT WATERMARK PATENT TRADE MARK ATTORNEYS P20693AU03 Associated Physical Media Submitted 11111Basic Document (ie Convent 11111Verified Translation [III]Description Z] Claims LI~ Abstract ~I~j Drawings EIII Gene Sequence Usting LII] CD-Rom or Diskette Other LIIJ (eg. Deeds, Assignrr ion/Priority Document) nents, etc.)