AU2006200800A1 - 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: r _l CORYNEBACTERIUM GLUTAMICUM GENES ENCODING PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM

PROTEINS

Related Applications 0 This application claims priority to U.S. Provisional Patent Application No.: 0 0 60/142,691, filed on July 1, 1999, and also to U.S. Provisional Patent Application No.: S60/150,310, filed on August 23, 1999, incorporated herein in their entirety by this \0 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.

C

I glutamicum 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

S

transformation. These novel nucleic acid molecules encode proteins, referred to herein C as phosphoenolpyruvate:sugar phosphotransferase system (PTS) proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonly used in O industry for the large-scale production of a variety of fine chemicals, and also for the 00 degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of Sterpenoids. The PTS nucleic acid molecules of the invention, therefore, can be used to IND 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 glutamicum 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 -3- (NI this microorganism. Given the availability of cloning vectors for use in SCorynebacterium 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 (C 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.

0 Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention 00 may be utilized in the genetic engineering of this organism to make it a better or more 0efficient producer of one or more fine chemicals.

\O 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 of metabolites 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 Sresponsive to such negative regulation, thereby permitting greater intracellular Sconcentrations of one or more sugars to be achieved, and, by extension, more 3 efficient production or greater yields of one or more fine chemicals from organisms containing such mutant PTS proteins.

C 5 This invention provides novel nucleic acid molecules which encode proteins, referred to herein as phosphoenolpyruvate:sugar phosphotransferase O system (PTS) proteins, which are capable of, for example, participating in the oo 0 import of high-energy carbon molecules glucose, fructose, or sucrose) into C 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 Corynebacterium glutamicum nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, or a complement thereof.

An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30, or a complement thereof.

An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30, or a complement thereof.

S* An isolated nucleic acid molecule comprising a nucleotide sequence c which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ C 5 ID NO:29, or a complement thereof.

An isolated nucleic acid molecule comprising a fragment of at least contiguous nucleotides of the nucleotide sequence of SEQ ID SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID (N NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, or a complement thereof.

An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least identical to the entire nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29.

An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence.

A host cell comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID N 5 SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID SEQ ID NO:27 and SEQ ID NO:29, wherein the nucleic acid 0 molecule is disrupted.

00 S* A host cell comprising a nucleic acid molecule selected from the N group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID 0 10 SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID c SEQ ID NO:27 and SEQ ID NO:29, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29.

A host cell comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID SEQ ID NO:27 and SEQ ID NO:29, wherein the regulatory region of the nucleic acid molecule is modified relative 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 acid fragments suitable as primers or hybridization probes for the detection or amplification 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 Sodd-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 c 5 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 00

IN

c-i C 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 CK1 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 O sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., 00 sufficiently homologous to an amino acid sequence of the invention such that the protein Sor portion thereof maintains a PTS activity. Preferably, the protein or portion thereof IN 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.

In another embodiment, the isolated nucleic acid molecule is at least Snucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention a sequence of an odd- Cl numbered SEQ ID NO in the Sequence Listing). Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, O the isolated nucleic acid encodes a naturally-occurring C. glutamicum PTS protein, or a 00 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 Corynebacterium 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 diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention the (1 sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 34)) in a subject, Sthereby 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 C,1 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 O carbon molecules glucose, fructose, or sucrose) into C. glutamicum, and also may 00 participate in one or more C. glutamicum intracellular signal transduction pathways. In another preferred embodiment, the isolated PTS protein or portion thereof is sufficiently IND 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. glulamicum intracellular signal transduction pathways, or has one or more of the activities set forth in Table 1.

(11 Alternatively, the isolated PTS protein can comprise an amino acid sequence Swhich is encoded by a nucleotide sequence which hybridizes, hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%, more C 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 O the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred 00 that the preferred forms of PTS proteins also have one or more of the PTS bioactivities described herein.

INDThe 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 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.

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 S 4 agent. In a preferred embodiment, the cell is modulated for the uptake of one or more Ssugars, 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 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 Ssmall molecules, active PTS proteins, and nucleic acids encoding PTS proteins that have 0 been introduced into the cell. Examples of agents which inhibit PTS activity or expression include small molecules, and antisense PTS nucleic acid molecules.

O 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 r, 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 1 explicated below.

1. Fine Chemicals 00 The term 'fine chemical' is art-recognized and includes molecules produced by San 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 et 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 L- C amino 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, SStryer, L. Biochemistry, 3 rd edition, pages 578-590 (1988)). The 'essential' amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, Sand 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.

-12-

C

N The biosynthesis of these natural amino acids in organisms capable of Sproducing 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.

Cl Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of aketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine Sare each subsequently produced from glutamate. The biosynthesis ofserine is a three- 00 step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and 0resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both IsO 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 11step pathway. Tyrosine may also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all biosynthetic products ofpyruvate, 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 ofhistidine 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.

SB. 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 00 they are readily synthesized by other organisms, such as bacteria. These molecules are Seither bioactive substances themselves, or are precursors of biologically active I 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).

-14r, Thiamin (vitamin BI) is produced by the chemical coupling of pyrimidine and Dthiazole moieties. Riboflavin (vitamin B 2 is synthesized from (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin C 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 00 the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic Sacid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-3-alanine) can be produced O 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 Bs), 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 12 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 r, known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are Salso termed 'niacin'. Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide C 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 large- 00 scale culture of microorganisms, such as riboflavin, Vitamin B 6 pantothenate, and Obiotin. Only Vitamin B 12 is produced solely by fermentation, due to the complexity of N 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 -16- (N1 anti-proliferants (Smith, (1995) "Enzymes in nucleotide synthesis." Curr. Opin.

SStruct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates C1 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 0 chemicals themselves, commonly used as flavor enhancers IMP or GMP) or for 00 several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and ORelated Compounds in Biotechnology vol. 6, Rehm et eds. VCH: Weinheim, p. 561- INO 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 phosphatc (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.

-17r, D. Trehalose Metabolism and Uses q. Trehalose consists of two glucose molecules, bound in a, a-1,1 linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen C 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.

O Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech. 16: 460- 0 0 467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and 0 Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from ^O 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 IIG 1 u for the Enzyme II complex which binds glucose). Enzymes II specific for mono-, di-, or oligosaccharides, like glucose, fructose, mannose, galactose, ribose, -18r, sorbose, ribulose, lactose, maltose, sucrose, raffinose, and others are known. Enzyme I Stransfers phosphoryl groups from phosphoenolpyruvate (PEP) to the phosphoryl carrier protein, HPr. HPr then transfers the phosphoryl groups to the different Enzyme II C 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 O structurally unrelated families. Further, the number and homology between these genes c vary from bacteria to bacteria. The E. coli genome encodes 38 different PTS proteins, O 33 of which are subunits belonging to 22 different transporters. The M. genitalium N 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 IIAG'IIICBGIc) or as domains of a single polypeptide chain IICBAGICNAc). 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 IIAG l c, and the activation of adenylate cyclase by the phosphorylated version of this protein. Also, the HPr and the IIB domains of some transporters in these microorganisms regulate gene expression by reversible -19phosphorylation of transcription antiterminators. In gram-positive bacteria, the activity 1 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 c1 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 Smovement of the bacterium (Sonenshein, A. et al., eds. Bacillus subtilis and other Sgram-positive bacteria. ASM: Washington, Neidhardt, et al., eds. (1996) SEscherichia coli and Salmonella. ASM Press: Washington, Lengeler et al., (1999).

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

III. 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. glufamicum 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 Sthe 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 O references pertaining to the PTS system, see: Stryer, L. (1988) Biochemistry. Chapter 00 37: "Membrane Transport", W.H. Freeman: New York, p. 959-961; Darnell, J. et al.

S(1990) Molecular Cell Biology Scientific American Books: New York, p. 552-553, and SMichal, 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 1. 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, -21then, 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 C 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 00 modulating the production of a desired molecule, such as a fine chemical, in a Smicroorganism such as C. glutamicum. Using recombinant genetic techniques, one or N 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 Corynebacterium glutamicum strain available through the American Type 22

C

Culture Collection, given designation ATCC 13032. The nucleotide sequence of the q. 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 C NOs and even-numbered SEQ ID NOs, respectively.

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

00 The present invention also pertains to proteins which have an amino acid Ssequence which is substantially homologous to an amino acid sequence of the invention \0 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 -23- C of sequence upstream from the 5' end of the coding region and at least about Snucleotides 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 Cdouble-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 O acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank 00 the nucleic acid sequences located at the 5' and 3' ends of the nucleic acid) in the Sgenomic DNA of the organism from which the nucleic acid is derived. For example, in \O 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- -24r thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) Sand DNA can be prepared using reverse transcriptase Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, 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 0 can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification I techniques. The nucleic acid so amplified can be cloned into an appropriate vector and 0 10 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 r, in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the Scorresponding 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 1 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 O molecules which they encode, such that they can be readily correlated. For example, the 00 amino acid sequences designated RXA01503, RXN01299, RXS00315, and RXC00953 Oare translations of the coding regions of the nucleotide sequence of nucleic acid ID 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 -26r, Listing the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize Sto 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 r,1 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%, O 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 00 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, S87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least IDabout 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. gluramicum 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 -27- C 1 sequences or naturally occurring mutants thereof. Primers based on a nucleotide Ssequence of the invention can be used in PCR reactions to clone PTS homologues.

SProbes 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 O group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- 00 factor. Such probes can be used as a part of a diagnostic test kit for identifying cells Swhich misexpress a PTS protein, such as by measuring a level ofa PTS-encoding IN 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 of high-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 4 In another embodiment, the protein is at least about 50-60%, preferably at least Sabout 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).

00 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, IDthe term "biologically active portion of a 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 -29- (1N glutamicum protein which is substantially homologous to an amino acid sequence of the Sinvention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing).

C1 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, O such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to 00 the present invention. In one embodiment, the invention includes nucleotide and amino Sacid sequences having a percent identity to a nucleotide or amino acid sequence of the IN 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 ,11 within a population due to natural variation. As used herein, the terms "gene" and S"recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a PTS protein, preferably a C. glutamicum PTS protein. Such natural r 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 O in PTS that are the result of natural variation and that do not alter the functional activity 00 of PTS proteins are intended to be within the scope of the invention.

SNucleic 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. glutamicum 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 -31r[ encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C.

Sglutamicum PTS protein.

In addition to naturally-occurring variants of the PTS sequence that may exist in r, 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 O changes in the amino acid sequence of the encoded PTS protein, without altering the 00 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 Snucleotide 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- -NI 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 C 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 0 0 sequence one of the amino acid sequences of the invention) is occupied by the Ssame amino acid residue or nucleotide as the corresponding position in the other I 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, glutamine, 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, phenylalanine, 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 Smutants 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 0 determined using, for example, assays described herein (see Example 8 of the Exemplification).

I In addition to the nucleic acid molecules encoding PTS proteins described above, 0 10 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 Santisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an C antisense nucleic acid an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to Sincrease the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, phosphorothioate Sderivatives and acridine substituted nucleotides can be used. Examples of modified O 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 r, 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 1 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 00 prokaryotic, viral, or eukaryotic promoter are preferred.

C In yet another embodiment, the antisense nucleic acid molecule of the invention I is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms 0 10 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 Haselhoffand 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 d 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 SAnother aspect of the invention pertains to vectors, preferably expression Svectors, 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 IN 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 ofplasmids. 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 -37- N host cell when the vector is introduced into the host cell). The term "regulatory Ssequence" is intended to include promoters, enhancers and other expression control elements polyadenylation signals). Such regulatory sequences are described, for rc example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which O direct constitutive expression of a nucleotide sequence in many types of host cell and 0 0 those which direct expression of the nucleotide sequence only in certain host cells.

SPreferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, 0 trp-tet-, Ipp-, lac-, Ipp-lac-, lacI q T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SP02, X-PRor 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, STLSI, 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. et 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 0 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 NO 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, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- IIII 13-B1, .gtl 1, pBdCl, and pET lid (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 -39r, transcription from a hybrid trp-lac fusion promoter. Target gene expression from the d) pET 1 d vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by

C

1 host strains BL21(DE3) or HMS 174(DE3) from a resident 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 Splasmids pIJ01, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for transformation Sof 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 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).

(NI 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.

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

O In another embodiment, the PTS proteins of the invention may be expressed in 00 unicellular plant cells (such as algae) or in plant cells from higher plants the Sspermatophytes, such as crop plants). Examples of plant expression vectors include I0 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 of T cell receptors (Winoto and -41r, Baltimore (1989) EMBO J. 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), 1 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 0 are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) 0 Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) NO 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 ofantisense 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- "1 A host cell can be any prokaryotic or eukaryotic cell. For example, a PTS Sprotein 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 C 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 O nucleic acid and protein molecules of the invention are set forth in Table 3.

00 Vector DNA can be introduced into prokaryotic or eukaryotic cells via Sconventional transformation or transfection techniques. As used herein, the terms IDO "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 -43r, homologue 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 O PTS gene is mutated or otherwise altered but still encodes functional protein the 00 upstream regulatory region can be altered to thereby alter the expression of the endogenous PTS protein). In the homologous recombination vector, the altered portion IND 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 amicroorganism. 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 -44- "11 more 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 O further provides methods for producing PTS proteins using the host cells of the 00 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 I:O 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 C N protein. In one embodiment, the language "substantially free of chemical precursors or Sother 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 Cabout 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, 00 isolated proteins or biologically active portions thereof lack contaminating proteins from Sthe same organism from which the PTS protein is derived. Typically, such proteins are IN 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. glutamicum, 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%, -46r, 499% 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 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 00 the PTS activities described herein. For example, a preferred PTS protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which IND 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 gluiamicum, 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 glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention.

-47- N 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 C 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 SPTS protein, and exhibit at least one activity of a PTS protein. Typically, biologically 0 0 active portions (peptides, peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, S37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with IN 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- -48r, 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 C 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 00 and/or secretion of a PTS protein can be increased through use of a heterologous signal sequence.

I 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 -49r, PTS protein and homologues thereof of the present invention may modulate the activity Sof one or more sugar transport pathways or intracellular signal transduction pathways in which PTS proteins play a role in this microorganism.

1 In an alternative embodiment, homologues of the PTS protein can be identified by screening combinatorial libraries of mutants, truncation mutants, of the PTS O protein for PTS protein agonist or antagonist activity. In one embodiment, a variegated 0 0 library of PTS variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of PTS variants O 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.

r, 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 C 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 O analysis, for screening large gene libraries typically include cloning the gene library into 00 replicable expression vectors, transforming appropriate cells with the resulting library of Ovectors, and expressing the combinatorial genes under conditions in which detection of a I 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 glulamicum 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 N under stringent conditions with a probe spanning a region of a C. glutamicum gene Swhich is unique to this organism, one can ascertain whether this organism is present.

SAlthough Corynebacterium glutamicum itself is nonpathogenic, it is related to Ci pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile 0 infection which involves both local and systemic pathology. In this disease, a local 00 lesion develops in the upper respiratory tract and involves necrotic injury to epithelial Scells; the bacilli secrete toxin which is disseminated through this lesion to distal LO 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 diphtheriae 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 ofC. 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 -52- FAlocalization 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 Sacid sequence to which the protein binds. Further, the nucleic acid molecules of the c 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 O map in related bacteria, such as Brevibacterium lactofermentum.

00 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 IDinvention 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- C 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.

SThis 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 00 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 Sproduction. 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 ofmutagenesis strategies for PTS proteins to result in increased yields of a desired compound is not meant to be limiting; variations on these -54- ,11 mutagenesis strategies will be readily apparent to one of ordinary skill in the art. By Sthese 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 CK1 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 O natural product of C. glutamicum, which includes the final products of biosynthesis 00 pathways and intermediates of naturally-occurring metabolic pathways, as well as Smolecules which do not naturally occur in the metabolism of C. glutamicum, but which Sare 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.

2006200800 24 Feb 2006 TABLE 1: Genes Included in the Invention PHOSPHOENOLPYRUVATE: SUGAR PHOSPHOTRANSFERASE SYSTEM Nucleotide Amino Acid Identification Co~nti NT Start NT Stop Function SEQIDO SEIDNO Code 12 RXS00315 PTS SYSTEM, SUCROSE-SPECIFIC IIABC COMPONENT (EIIABC-SCR) (SUCROSE- PERMEASE IIABC COMPONENT(PHOSPHOTRANSFERASE ENZYME 11, ABC COMPONENT) (EC 2.7.1.69) 3 4 F RXA00315 GR00053 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 RXA01503 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 WV0068 11954 9891 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 9 10 F RXA01 299 GR00375 6 446 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 RXA01889 GR00540 77 631 PTS SYSTEM, FRUCTOSE-SPECIFIC IIBC COMPONENT (EC 2.7.1.69) 16 RXAOO951 GR00261 564 172 PTS SYSTEM, MANNITOL (CRYPTIC) -SPECIFIC ILA COMPONENT (EIIA-(C)MTL) (MANNITOL (CRYPTIC)- PERMEASE IA COMPONENT) (PHOSPHOTRANSFERASE ENZYME 11, A COMPONENT) (EC 2.7.1.69) 17 18 RXN01244 WV0068 14141 15844 PHOSPHOENOLPYRUVATE-PROTEIN PHOSPHOTRANSFERASE (EC 2.7.3.9) 19 20 F RXA01 244 GR00359 4837 3329 PH-OSPHOENOLPYRUVATE-PROTEIN PH-OSPHOTRANSFERASE (EC 2.7.3.9) 21 22 RXA01300 GR00375 637 903 PHOSPHOCARRIER PROTEIN HPR 23 24 RXN03002 WV0236 1437 1844 PTS SYSTEM. MANNITOL (CRYPTIC) -SPECIFIC IIA COMPONENT (EIIA-(C)MTL) (MANNITOL (CRYPTIC)-PERMEASE IIA COMPONENT) (PHOSPH-OTRANSFERASE ENZYME 11, A COMPONENT) (EC 2.7.1.69) 26 RXCO0953 WV0260 1834 1082 Membrane Spanning Protein involved in PTS system 27 28 RXC03001 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 CR00642 3395 4633 PHOSPH-OENOLPYRUVATE SUGAR PHOSPHOTRANSFERASE 33 34 F RXA01943 GR00557 3944 3540 err gene; phosphotransferase system glucose-specific enzyme III 2006200800 24 Feb 2006 2 Excluded Genes GenBank~m 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 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, 9519442-A 5 07/20/95 A45585 A45587 AB0O3 132 murC; ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and characterization of the fisZ gene from coryneform bacteria," Biochem. Biophys. Res. Commun., 236(2):383-388 (1997) ABO 15023 murC; ftsQ Wachi, M. et al. "A murC gene from Coryneform bacteria," App. Microbil.

Biotechnol., 51 (2):223-228 (1999) ABO 18530 dtsR Kimura, E. et al. "Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibact'erium _____________lactofermenturn," Biosci. Biotechnol Biochem., 60(10): 1565-1570 (1996) AB018S31 _dtsRl; dtsR2 AB020624 muri D-glutamate racemase AB023377 tkt transketolase AB024708 gltB; gltD Glutamine 2-oxoglutarate aminotransferase large and small subunits AB025424 acn aconitase AB027714 rep Replication protein AB027715 rep; aad Replication protein; aminoglycoside adenyltransferase AF005242 argC dehydrogenase AF005635 glnA Glutamine synthetase AF030405 hisF cyclase AF030520 argG Argininosuccinate synthetase AF03 1518 argF Omnithine carbamolytransferase AF036932 aroD 3-dehydroquinate dehydratase AF038548 pyc IPyruvate carboxylase 2006200800 24 Feb 2006 Table 2 (continued) AF038651 dciAE; apt; ret Dipeptide-binding protein; adenine Wehmeier, L. et at. "The role of the Corynebacterium glutamicum rel gene in phosphoribosyltransferase; GTP (p)ppGpp metabolism," Microbiology, 144:1853-1862 (1998) pyrophosphokinase AF04 1436 argk Arginine repressor AF045998 imnpA Inositot monophosphate phosphatase AF048764 argH Argininosuccinate tyase AF049897 argC; argi; argB; N-acetytgtutamylphosphate reductase; argD; argF; argR; ornithine acetyltransferase; NargG; argH acetyiglutamate kinase; acetylomnithine transminase; ornithine carbamoyltransferase; arginime repressor; argininosuccinate synthase;I ~~argininosuccinate lyase AF0501t09 inhA Enoyt-acyt carrier protein reductase AF050 166 hisG ATP phosphoribosyltransferase AF051t846 hisA Phosphoribosytform imino-5-amino- I1phosphoribosyl-4-imidazotecarboxamide isomerase AF052652 metA Homoserine 0-acetyltransferase Park, S. et al. "Isotation and anatysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in Corynebacteriumn glutamicum," Mo!.

8(3):286-294 (1998) AF053071 aroB Dehydroquinate synthetase AF060558 hisH Gtutamine amidotransferase AF086704 hisE Phosphoribosyt-ATP- _____________pyrophosphohydrotase AFt 14233 aroA 5-enotpyruvytshikimate 3-phosphate A F 16184 panD L-aspartate-alpha-decarboxy lase precursor Dusch, N. et at. "Expression of the Corynebacterium gtutamicumn panD gene encoding L-aspartate-atpha-decarboxy lase leads to pantothenate overproduction in Escherichia coti," App. Environ. Microbial., 65(4)[530- 1539 (1999) AF124518 aroD; aroE 3-dehydroquinase; shikimate dehydrogenase AF124600 aroC; aroK; aroB; Chorismate synthase; shikimate kinase; 3pepQ dehydroquinate synthase; putative cytoplasmic peptidase AF145897 inhA AF145898 inhA 2006200800 24 Feb 2006 Table 2 (continued) AJOO 1436 ectP Transport of ectoine, glycine betaine, Peter, H. et al. "Corynebacterium glutamnicumn is equipped with four secondary proline carriers for compatible solutes: Identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP," J. Bacterial, 180(22):6005-6012 (1998) AJ004934 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(1 2):3 159-3165 (1998) AJO07732 ppc; secG; amt; ocd; Phosphoenolpyruvate-carboxylase; high soxA affinity ammonium uptake protein; putative om ithine-cyclodecarboxylase; sarcosine oxidase AJO103 19 NiY, 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 enzmnye); signal recognition particle;, low proteins," FEMS Microbial. 173(2):303-310 (1999) affinity ammonium uptake AJ132968 cat Chloramphenicol 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. Biochein., 254(2):395-403 (1998) AJ238250 ndh NADH dehydrogenase AJ238703 porA Porin Lichtinger, T. et al. "Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum: The channel is formned by a low mass polypeptide," Biochemistry, 37(43): 15024-15032 (1998) D1 7429 Transposable element IS3 1831 Vertes et al."lsolation and characterization of IS3 183 1, a transposable element from Corynebacteriumn glutamicum," Mol. MicrobioL., I11(4):739-746 (1994) D84 102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al. "Molecular cloning of the Corynebacteriumn glutamicumn (Brevibacterium lactofermentumn AJ 12036) odhA gene encoding a novel type 2-oxoglutarate dehydrogenase," Microbiology, 142:3347-3354 (1996) E01358 hdh; hk Homoserine dehydrogenase; homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: IP_ kinase 1987232392-A 1 10/12/87 E0 1359 Upstream of the start codon of homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: JP gene 1987232392-A 2 10/12/87 E0 1375 Tryptophan operon E01376 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 I 10/24/87 2006200800 24 Feb 2006 Table 2 (continued) E01377 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 tryptophan," Patent: JP 1987244382-A 1 10/24/87 E03937 Biotin-synthase Hatakeyama, K. et al. "DNA fragment containing gene capable of coding biotin synthetase and its utilization," Patent: JP 1992278088-A t 10/02/92 E04040 Diamino pelargonic acid aminotransferase Kohama, K. et al. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A I 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: JP 1993030977-A 1 02/09/93 E04376 Isocitric acid lyase Katsumata, 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 1993056782-A 3 03/09/93 E04484 Prephenate dehydratase Sotouchi, N. et al. "Production of L-phenylalanine by fermentation," Patent: JP 1993076352-A 2 03/30/93 E05108 Aspartokinase Fugono, N. et al. "Gene DNA coding Aspartokinase and its use," Patent: JP 1993184366-A 1 07/27193 E051 12 Dihydro-dipichorinate synthetase Hatakeyama, K. et al. "Gene DNA coding dihydrodipicolinic acid synthetase and its use," Patent: JP 1993184371 -A 1 07/27/93 E05776 Diaminopimelic acid dehydrogenase Kobayashi, M. et al. "Gene DNA coding Diaminopimelic acid dehydrogenase its use," Patent: J P 1993284970-A 1 11/02/93 E05779 Threonine synthase Kohama, K. et al. "Gene DNA coding threonine synthase and its use," Patent: JP 1993284972-A 1 11/02/93 E061 10 Prephenate dehydratase Kikuchi, T. et al. "Production of L-phenylalanine by fermentation method," Patent: JP 199334488 1-A 1 12/27/93 E061 11 Mutated Prephenate dehydratase Kikuchi, T. et al. "Production of L-phenylalanine by fermentation method," JP 199334488 1-A 1 12/27/93 E06 146 Acetohydroxy acid synthetase Inui, M. et al. "Gene capable of coding Acetohydroxy acid synthecase and its Patent: JP 1993344893-A 1 12/27/93 E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A 1 E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A 1 2006200800 24 Feb 2006 Table 2 (continued) E06827 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A 1 03/08/94 E07701 secY Honno, N. et al. "Gene DNA participating in integration of membraneous to membrane," Patent: JP 1994 169780-A 1 06/21/94 E08 177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08 178, Feedback inh ibition-re leased Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from E08179, feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08 180, E08 181, E08 182 E08232 Acetohydroxy-acid isomeroreductase lnui, M. et al. "Gene DNA coding acetohydroxy acid isomeroreductase," 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 H-atakeyama, K. et al. "DNA fragment having promoter function in promoter region coryneform bacterium," Patent: JP 1995031476-A 1 02/03/95 E08646 Biotin synthetase .Hatakeyama, K. et al. "DNA fragment having promoter function in coryneform bacterium," Patent: JP 1995031l476-A 1 02/03/95 E08649 Aspartase Kohama, K. et al "DNA fragment having promoter function in coryneform Patent: JP 1995031478-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 Diaminopimelic acid decarboxylase Madori, M. et al. "DNA fragment containing gene coding Diaminopimelic acid decarboxylase and utilization thereof," Patent: JP 1995075579-A 1 03/20/95 E 12594 Serine hydroxymethyltransferase l-atakeyama, K. et al. "Production of L-trypophan," Patent: JP 199702839 1-A 1 02/04/97 1312760, transposase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: E12759, JP 199707029 1-A 03/18/97 E12764 Arginyl-tRNA synthetase; diaminopimelic Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: acid decarboxylase JP 1997070291 -A 03/18/97 E12767 Dihydrodipicolinic acid synthetase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 E12770 aspartokinase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 El27 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: 1997070291-A 03/18/97 2006200800 24 Feb 2006 2 (continued) E13655 Glucose-6-phosphate dehydrogenase l-atakeyania, K. et al. "Glucose-6-phosphate dehydrogenase and DNA capable of coding the same," Patent: JP 1997224661 -A 1 09/02/97 L01508 livA Threonine dehydratase Moeckel, B. et al. "Functional and structural analysis of the threonine dehydratase of Corynebacteriurn glutamicum," J Bacterial., 174:8065-8072 (1992) 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-arabi noheptu losonate-7-phosph ate synthase gene," FEMS MicrobiaL Let., 107:223-230 (1993) L09232 IlvB; ilvN; ilvO 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-ilvC operon," J Bacteriol, 175(17):5595- ___________Acetohydroxy acid isomeroreductase 5603 (1993) L18874 PIsM 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 11 and analyses of the deduced protein FEMS MicrobiaL Lett., 1 19(1-2):137-145 (1994) L27 123 aceB Malate synthase Lee, H-S. et al. "Molecular characterization of aceB, a gene encoding malate synthase in Corynebacterium glutamicum," J Microbial BiotechnaL, 4(4):256-263 (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 (1994) 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) *M16175 5S rRNA Park, Y-H. et al. "Phylogenetic analysis of the coryneform bacteria by 56 rRN'A sequences," J Bacterial., 169:1801-1806 (1987) *M16663 trpE Anthranilate synthase, 5' end Sano, K. et al. "Structure and function of the trp, operon control regions of Brevibacterium lactofermentumn, a glutamic-acid-producing bacterium," Gene, 52:191-200 (1987) 16664 trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and function of the trp operon conftrol regions of Brevibacterium lactofermentumn, a glutamic-acid-producing bacterium," Gene, 1 52:191-200 (1987) 2006200800 24 Feb 2006 2 (continued) M25819 Phosphoenolpyruvate carboxylase O'Regan, M. et al. "Cloning and nucleotide sequence of the Phosphoenolpymuvate carboxylase-coding gene of Corynebacterium glutamicum ATCC 13032," Gene, 77(2):237-251 (1989) 106 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.

138:1167-1175 (1992) 107, 23S rRINA gene insertion sequence Roller, C. et al. "Gram-positive bacteria with a high DNA G+C content are 108 characterized by a common insertion within their 23S rRNA genes," J Gen.

icrobial, 138:1167-1175 (1992).

M89931I aecD; bmQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, 1. et at. "The Corynebacterium glutam icum aecD gene encodes a C-S uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination activity that degrades aminoethylcysteine," J Bacterial., 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in Corynebacteriumn glutamicum ATCC 13032 is directed by the brnQ gene product," Arch Microbial, 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," Appi. Environ. Microbial. 59(3):791-799 (1993) UI 11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican, L.K. (1994) Complete nucleotidle sequence of the Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology University College Galway, Ireland.

U 13922 cglIM; cglIR; ciglIR Putative type 11 5-cytosoine Schafer, A. et at. "Cloning and characterization of a DNA region encoding a m ethyltransfe rase; putative type 11 stress-sensitive restriction system from Corynebacterium glutamicum ATCC restriction endonuclease; putative type I or 13032 and analysis of its role in intergeneric conjugation with Eseherichia type Ill restriction endonuclease coli," J Bacterial., 176(23):7309-7319 (1994); Schafer, A. et at. "The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine in an McrBCdeficient Escherichia coli strain," Gene, 203(2):95-101 (1997) U 14965 U31224 ppx Ankri, S. et at. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J1 Bacterial, 178(15):4412-4419 (1996) U3 1225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacteriumn glutamnicumproline biosynthetic pathway: A natural bypass of the proA step," J Bacterial., 178(15):4412-4419 (1996) U31230 obg; proB; unkdh ?;gamma glutamyl kinase;similar to D- Ankri, S. et at. "Mutations in the Corynebacterium glutamicumproline isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the proA step," J Bacterial, _____________dehydrogenases 178(15):4412-4419 (1996) 2006200800 24 Feb 2006 Table2 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 glutamicum," Gene. 175:15-22 (1996) U35023 thtR;, accBC Thiosulfate sulf'urtransferase;I acyl CoA Jager, W. et al. "A Corynebacteriumn glutamicumn gene encoding a two-domain carboxylase protein similar to biotin carboxylases and biotin-carboxyl-carr ier proteins," Microbial., 1 66(2);76-82 (1996) U43535 cmr Multidrug resistance protein Jager, W. et al. "A Corynebacterium glutamicum gene conferr ing multidrug resistance in the heterologous host Escherichia coli," J Bacterial., 179(7):2449-2451 (1997) U43536 clpB Heat shock ATP-binding protein U53587 aphA-3 3'5' -aminoglycoside phosphotransferase U89648 Corynebacteriumn glutamicum unidentified sequence involved in histidine biosynthesis, partial sequence X04960 trpA; trpB; trpC; trpD; Tryptophan operon Matsui, K. et al. "Complete nucleotide and deduced amino acid sequences of trpE; trpG; trpL the Brevibacterium lactofiermentumn tryptophan operon," Nucleic Acids Res., 14(24):101 13-10114 (1986) X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al. "Nucleic sequence of the lysA gene of Corynebacteriumn decarboxylase, EC 4.1.1.20) glutamicum and possible mechanisms for modulation of its expression," Mal.

Gen. Genet., 212(l):1 12-119 (1988) X 14234 EC 4.1.1.31 Phosphoenolpyruvate carboxylase Eikmanns, B.J. et al. "The Phosphoenolpyruvate carboxylase gene of Corynebacteriumn glutamicum: Molecular cloning, nucleotide sequence, and expression," Ma!. Gen. Genet., 218(2):330-339 (1989); Lepiniec, L. et al.

"Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function and molecular evolution," Plan. Mal. Bial., 21 (3):487-502 (1993) X173 13 fda Fructose-bisphosphate aldolase Von der Osten, C.H. et al. "Molecular cloning, nucleotide sequence and finestructural analysis of the Corynebacterium glutamicumn fda gene: structural comparison of C. glutamnicumn fructose-I1, 6-biphosphate aldolase to class I and class 11 aldolases," Ma!. Microbial., X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al. "Nucleic sequence of the dapA gene from Corynebacterium glutamicum," Nucleic Acids Res., 18(21):6421 (1990) X54223 AttB-related site Cianciotto, N. et al. "DNA sequence homology between att B3-related sites of Corynebacterium diphtheriae, Corynebacteriumn ulcerans, Corynebacterium glutamicumn and the attP? site of lambdacorynephage," FEMS. Microbial, 66:299-302 (1990) X54740 argS; lysA Arginyl-tRNA synthetase; Diamninopimelate Marcel, T. et al. "Nucleotide sequence and organization of the upstream region decarboxylase of the Corynebacterium glutamicumn lysA gene," Mo. Microbial., 4(1 1):1819- 1830(1990) 2006200800 24 Feb 2006 2 c ontinued) X55994 trpL; trpE Putative leader peptide; anthranilate Heery, D.M. et al. "Nucleotide sequence of the Corynebacterium glutamicum synthase component I trpE gene," Nucleic Acids Res., I18(23):7 13 8(1990) X56037 thrC Threonine synthase Han, K.S. et al. "The molecular structure of the Corynebacterium glutamicum threonine synthase gene," Mo!. Microbial, 4(10):1693-1702 (1990) X56075 attB-related site Attachment site Cianciotto, N. et al. "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) X57226 IysC-alpha; lysC-beta;, Aspartokinase-aipha subunit; Kalinowski, J. et al. "Genetic and biochemical analysis of tihe Aspartokinase asd Aspartokinase-beta subunit; aspartate beta from Corynebacterium glutamicum," Mo. Microbial., 5(5):1 197-1204 (1991); semnialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase genes IysC alpha and IysC beta overlap and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in ___________Corynebacterium glutamicum," Gen. Genet., 224(3):317-324 (1990) X59403 gap;pgk; Ipi G lyceraldehyde-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 glyceraldehyde-3 -phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomeras," J. Bacterial, 174(19):6076-6086 X59404 gdh Glutamate dehydrogenase Bormann, E.R. et al. "Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase," Mal Micrabiol, 6(3):3 17-326 X60312 lysi L-lysine permease Seep-Feldhaus, A.H. et al. "Molecular analysis of the Corynebacterium glutamicum lysi gene involved in lysine uptake," Mal. Microbial, 5(12):2995- 3005 (1991) X66078 copi Ps I protein Joliff, G. et al. "Cloning and nucleotide sequence of the cspl gene encoding PSI, one of the two major secreted proteins of Corynebacterium glutamicum: The deduced N-terminal region of PSI is similar to the Mycobacterium antigen complex," Ma!. Mlicrobial, 6(16):2349-2362 (1992) X661 12 glt Citrate synthase Eikmanns, B.J. et al. "Cloning sequence, expression and transcriptional analysis of the Corynebacterium glutamicum g~tA gene encoding citrate synthase," MicrobiaL., 140:1817-1828 (1994) X67737 dapB 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," Mo. Micrabial., 9(1):97-109 (1993) X69 104 153 related insertion element Bonamy, C. et al. "Identification of ISI1206, a Corynebacterium glutamicum 153-related insertion sequence and phylogenetic analysis," Ma!. Microbial, (1994) 2006200800 24 Feb 2006 Table 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 App!. Environ. Microbial, 60(l):133-140 (1994) X71489 icd Isocitrate dehydrogenase (NADP+) Eikmanns, B.J. et al. "Cloning sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme," J Bacterial, (1995) X72855 GDHA Glutamnate dehydrogenase (NADP+) X75083, mtrA 5-methyltryptophan resistance Heery, D.M. et al. "A sequence from a tryptophan-hyperproducing strain of X70584 Corynebacterium glutamicum encoding resistance to Biophys. Res. Cominun., 201(3):1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA mutant strains of Corynebacterium glutam icum and Brevibacterium lactofermentum," AppI.

Microbial. Biotechnal., 42(4):575-580 (1994) X75504 aceA; thiX Partial Isocitrate lyase; Reinscheid, D.i. et al. "Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme," J BacteriaL, t176(1 2):3474-3483 (1994) X76875 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," Antonie Van Leeuwenhoek, 64:285-305 (1993) X77034 tuf Elongation factor Tu Ludwig, W. et al. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit Anionic Van Leeuivenhoek~ 64:285-305 (1993) X77384 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 Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase: sequence analysis," Microbiology, 140:3099-3108 (1994) 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., 141:523-528 X81 191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al. "Structure of the gluABCD cluster encoding the gluD glutamate uptake system of Corynebacterium glutamicum," J Bacterial., 152-1158 (1995) X81379 dapE Succi nyld iam inop ime late desuccinylase Wehrmann, A. et al. "Analysis of different DNA fragments of Corynebacterium glutamicum complementing dapE of Escherichia cali," 40:3349-56 (1994) 2006200800 24 Feb 2006 2 (continued) X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al. "Phylogeny of the genus Corynebacteriumn deduced from analyses of small-subunit ribosomal DNA sequences," Int. J. Sys. Bacteriol., (1995) X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; Serebrijski, 1. et al. "Multicopy suppression by asd gene and osmotic stressdependent complemeniation 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 Bacterial., 1 77(24):7255-7260 (1995) X84257 16S rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of the genus Corynebacteriumn based on 16S rRNA gene sequences," In. J Syst. Bacterial., 45(4):724-728 (1995) X85965 aroP; dapE Aromatic amino acid permease; Wehrmann et al. "Functional analysis of sequences adjacent to dapE of C.

glutamicumn proline reveals the presence of aroP, which encodes the aromatic amino acid transporter," J Bacleriol., 1 77(20):5991-5993 (1995) X86 157 argB; argC; argD;, Acetylglutamnate 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; ornithine steps of the arginine pathway," Microbiology, 142:99-108 (1996) carbamnoyltransferase; glutamate Nacetyltransferase X89084 pta; ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D.J. et al. "Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotran sacety lase and acetate kinase," Microbiology, 145:503-513 (1999) X89850 attB 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 (1996) X90356 Promoter fragment FlI Patek, M. et al. "Promoters from Corynebacteriumn glutanhicum: cloning, molecular analysis and search for a consensus motif," Microbiology (1996) X90357 Promoter fragment F2 Patek, M. et al. "Promoters from Corynebacterium glutanticum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90358 Promoter fragment FI10 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90359 Promoter fragment F13 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) 2006200800 24 Feb 2006 Table 2 (continued) X90360 Promoter fragment P22 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90361 Promoter fragment F34 Patek, M. et al. "Promoters from Corynehacterium 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 and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90363 Promoter fragment P45 Patek, M. et al. "Promoters from Corynebacterium glutaniicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90364 Promoter fragment F64 Patek, M. et al. "Promoters from Corynebacterium glutainicum: 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 glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90366 Promoter fragment PF 10I Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology 142:1297-1309 (1996) X90367 Promoter fragment PF104 Patek, M. et al. "Promoters from Corynebacterium glutarnicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90368 Promoter fragment PPF109 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X935 13 amt Ammonium transport system Siewe, R.M. et al. "Functional and genetic characterization of the (methyl) ammonium uptake carrier of Corynebacterium glutamicum," J. Biol. Chem., (1996) X93514 betP Glycine betaine transport system Peter, H. et al. "Isolation, characterization, and expression of the Corynebacterium glutamnicuin betP gene, encoding the transport system for the solute glycine betaine," J. Bacteriol., 178(17):5229-5234 (1996) X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of the dapB-ORF2dapA-ORP4 operon of Corynebacterium glutamicum, encoding two enzymes involved in L-lysine synthesis," Biofechnol. Let., 19:1113-1117 (1997) X96471 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-lysine export from Corynebacteriuin glutamicum," Mo.

22(5):815-826 (1996) 2006200800 24 Feb 2006 Table 2 (continued) X96580 panB; panC; xyIB 3-methyl-2-oxobutanoate Sahm, H. et "D-pantothenate synthesis in Corynebacterium glutamicum and hydroxymethyltransf'erase; pantoate-beta- use of panBC and genes encoding L-valine synthesis for D-pantothenate alanine ligase; xylulokinase overproduction," App!. 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 lactoferinentum ___________(Corynebacterium glutamicum ATCC 13869)," Gene, 198:217-222 (1997) Y00 140 thrB Homoserine kinase Mateos, L.M. et al. "Nucleotide sequence of the homoserine kinase (thrB) gene of the Brevibacterium Iactoferrnentum," Nucleic Acids Res., 15(9):3922 (1987) YOOI151 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., 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 hom; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al. "Nucleotide sequence and fine structural analysis of the kinase Corynebacterium glutamicum hom-thrB operon," Mo!. Microbiol., 2(l):63-72 Y08964 murC; ftsQ/divD; ftsZ UPD-N-acetylmuramate-alanine ligase; Honrubia, M.P. et al. "Identification, characterization, and chromosomal division initiation protein or cell division organization of the ftsZ gene from Brevibacterium lactofermentum," Mo!. 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. Microbiol., 168(2):143-151 (1997) Y09548 pyc Pyruvate carboxylase Peters- Wend isch, P.G. et al. "Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene," Microbiology, 144:915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et al. "Analysis of the leuB gene from Corynebacterium App!. Microbiol. Biotechnol., 50(l):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," Microbial., 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 prolinefectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP," J Baclerial., I180(22):6005-6012 (1998) 2006200800 24 Feb 2006 2 (continued) Y13221 ginA Glutamine synthetase I Jakoby, M. et al. "Isolation of Corynebacteriumn glutamicumn glnA gene glutamine synthetase FEMS Microbiol Left, 154(l):81-88 (1997) Y 16642 lpd Dihydrolipoamide Y 18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis of the integration fu~nctions of φ304L: An module among corynephages," Virology, 255(1):150-159 (1999) Z21501 argS; lysA Arginyl-tRNA synthetase; diamninopimelate Oguiza, J.A. et al. "A gene encoding arginyl-tRNA synthetase is located in the decarboxylase (partial) upstream region of the lysA gene in Brevibacteriumn lactofermentum: Regulation of argS-lysA cluster expression by arginine," J Bacterial, 1 75(22):73 56-7362 (1993) Z21502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et al. "A cluster of three genes (dapA, orf2, and dapB) of dihydrodipicolinate reductase Brevibacteriumn lactofiermentumn encodes dihydrodipicolinate reductase, and a third polypeptide of unknown function," J Bacteriol, 175(9):2743-2749 (1993) Z29563 thrC Threonine synthase Malumbres, M. et al. "Analysis and expression of the thrC gene of the encoded threonine synthase," Appi. Environ. Microbial, 60(7)2209.22 19 (1994) Z46753 16S rDNA Gene for 16S ribosomal RNA Z49822 sigA SigA sigma factor Oguiza, J.A. et al "Multiple sigma factor genes in Brevibacterium lactofermnentum: Characterization of sigA and sigB," J1 BacierioL, 178(2):550- (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 gene," Gene 177:.103-107 (1996)- Z49824 orflI; sigB SigB sigma factor Oguiza, J.A. et al "Multiple sigma factor genes in Brevibacteriumn lactofermentum: Characterization of sigA and sigB," J Bacteriol, 178(2):550- (1996) Z66534 Transposase Correia, A. et al. "Cloning and characterization of an IS-like element present in the genome of Brevibacteriumn lactofermientumn ATCC 13869," Gene, rA 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 Brevibacteriuni Strains Which May be Used in the Practice of the Invention Brevibacterium ammoniagenes 21054 Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium ammaniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518 Brevibacterium fiavum B 11474 Brevibacterium flavum Bi 1472 Brevibacterium flavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brevibacterium flavum 21475 Brevibacterium tiavum 21517 Brevibacterium flavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum BI 1477 Brevibacterium flavum BI 11478 Brevibacterium flavum 21127 Brevibacterium flavum B1 1474 Brevibacterium healii 15527 Brevibacterium ketoglutamicum 21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevibacterium lactofermentum Brevibacterium lactofermentum 74 Brevibacterium lactofermentum 7 Brevibacterium lactofermentum 21798 Brevibacterium lactofermentum 21799 Brevibacterium lactofermnentum 21800 Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B 1470 Brevibacterium lactofermentum B 11471 Brevibacterium lactofermentumn 21086 Brevibacterium lactofermentumn 21420 Brevibacterium lactofermentum 21086 Brevibacterium lactofermentumn 31269 Brevibacterium linens 9174 Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacterium paraffinolyticum 11160 Brevibacterium Spec. ____717.73 Brevibacterium Spec. Brevibacterium Spec. 14604 Brevibacterium Spec. 21860 Brevibacterium Spec. 21864 Brevibacterium Spec. 21865 Brevibacterium Spec. 21866 Brevibacterium Spec. 19240 Corynebacterium acetoacidophilurn 21476 Corynebacterium acetoacidophilumn 13870 Corynebacterium acetoglutamicum BI 11473 Corynebacterium acetoglutamnicumn B11475 Corynebacterium acetoglutamnicumn 15806 Corynebacterium acetoglutarnicumn 21491 Corynebacterium acetoglutamnicumn 31270 Corynebacterium acetophilumn B3671I Corynebacterium ammoniagenes 6872 -2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacterium glutamnicumn 14067 Corynebacterium glutamnicumn 39137 Corynebacterium glutamicum 21254 Corynebacterium glutamnicumn 21255 Corynebacterium glutamnicumn 31830 Corynebacterium glutamnicumn 13032 Corynebacterium glutamnicumn 14305 Corynebacterium glutamicumn 15455___ Corynebacterium glutamnicumn 13058 Corynebacterium glutarnicumn 13059 Corynebacterium glutarnicum 13060 Corynebacterium glutamnicumn 21492 Corynebacterium glutamnicun 21513 Corynebacterium glutamnicumn 21526 Corynebacterium glutamnicumn 21543 Corynebacterium glutarnicumn 13287 Corynebacterium glutarnicumn 21851 Corynebacterium glutamnicumn 21253 Corynebacterium glutamicumn 21514 Corynebacterium glutarnicumn 21516 Corynebacterium glutamnicumn 21299 Corynebacterium Iglutamnicum 21300 72 Corynebacteri~um glutamicum 39684 Corynebacterium glutamicum 21488 Corynebacterium glutamicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 19223 Coz-ynebacterium glutamicum 13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum 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 glutamicum 21 568 Corynebacterium glutarbicum 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 glutamicuin 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum 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 B8182 Corynebacterium glutamicum B12416 Corynebacterium glutamicum 12417____ Corynebacterium glutamicum B12418 lCorynebacterium glutamicum BI 11476 73 Corynebacterium glutamicum 21608____ Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 11594 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 Schimnmelcultures, Baarn, NL NCTC: National Collection of Type Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen und Zelikulturen, Briunschweig, Germnany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (4hI edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.

2006200800 24 Feb 2006 TABLE 4: ALIGNMENT RESULTS Length Accession Name of Genbank Hit ID length Genbank Hit

(NT)

Source of Genbank Hit Date of homoloav Deooslt

(GAP)

1527 GB_BA1:AB007125 GBIN1:CELC47D2 GBHTG2:AC006732 rxa0l 503 372 GB PR3:AC00501 9 GBGSS12:AQ390040 GBGSS5:AQ784231.

rxa01299 2187 GBEST38:AW047296 GB_RO:AB004056 GBRQ:AB004056 416 GBBA1:SCJ21 GBV:MCU68299 4078 ABOO71 25 Serratia marcescens slaA gene for surface layer protein, complete Serratia marcescens 40,386 cds, isolate 8000.

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

188362 AC005019 Homo sapiens BAC clone GS250Al16 from 7p21 -p22, complete Homo sapiens 680 AQ390040 RPCII11-157C9.TJ RPCI-11 Homo sapiens genomic clone RPCI- H-omo sapiens 1 1-1 57C9.genomic survey sequence.

542 AQ784231 HS_3087_Bi_C1OIT7C CIT Approved Human Genomic Sperm Homo sapiens Library D Homo sapiens genomic clone Plate=3087 Col=19 Row=F, genomic survey sequence.

614 AW047296 UI-M.B1.amh-e-03-0-Ul-sl NIHBMAPMS2 Mus mtisculus Mus musculus.

cONA clone UI-M-BH-1-amh-e-03-0-UI mRNA sequence.

1581 AB004056 Rattus norvegicus mRNA for BarH-class homeodomain Ratlus norvegi transcription factor, complete cds.

1581 ABO04056 Rattus norvegicus mRNA for BarH-class homeodomain Rattus norvegi transcription factor, complete cds.

31717 AL109747 Streptomyces coelicolor cosmid J21. Slreptomyces 26-MAR-1998 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 18-Sep-99 41,031 2-Sep-98 40.717 2-Sep-98 :us :us coelicolor 34,913 5-Aug-99 230278 A3(2) U68299 Mouse cytomnegalovirus 1 complete genomic sequence. Mouse cytomnegalovirus 1 40,097 04-DEC-1 996 GBVIU93872 rxa01244 1827 GBBA1:AFAPHBI GBPR3:H5J836E13 GBE5T24:A1170227 =x0l 300 390 GB_PR3:HUMDODDA GBPAT:140899 GB PAT:140900 rxa00953 789 GB-BAI:SCJ2I GBBA1:BLTRP 133661 U93872 Kaposi's sarcoma-associated herpesvirus glycoprotein M, DNA Kaposi's sarcomareplication protein, glycoprolein DNA replication protein, FLICE associated herpesvirus inhibitory protein and v-cyclin genes, complete cds, and tegument 4501 M69036 Alcaligenes eutroplius protein H (phbH) and protein I (phbl) Ralstonia eutropha genes, complete cds.

78055 AL050326 Human DNA sequence fromn clone 836E13 on chromosome 20 Homo sapiens Contains ESTs, STS and GSSs, complete sequence.

409 Al 170227 EST216152 Normalized rat lung, Bento Scares Rattus sp. cONA Rattus sp.

clone RLUCF56 3Send, mRNA sequence.

26784 L39874 Homo sapiens deoxycytidylate deaminase gene, complete cds. Homo sapiens 26764 140899 Sequence 1 from patent US 5622851. Unknown.

1317 140900 Sequence 2 from patent US 5622851. Unknown.

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 1 3-MAY-1997 37,644 13-MAY-1997 39,398 5-Aug-99 39,610 10-Feb-99 46,753 29-Sep-97 r 7725 X04960 Brevibacterium lactofermenlumn tryptophan operon.

A3(2) Corynebactedrn glutamicumn Corynebactedrlu glutamicumn GBPAT:E01375 7726 E01 375 DNA sequence of tryptophan operon.

2006200800 24 Feb 2006 Table 4 (continued) rxa01943 2172 GBBAI:CORPTSMA 2656 118874 Corynebacterium glutamicumn phosphoenolpyruvate sugar phosphotransferase (ptsM) mRNA, complete cds.

GB,8A1 :BRLPTSG 3163 L18875 Brevibacterium lactafermentumn phosphoenolpyruvate sugar phosphotransferase (ptsG) gene, complete cds.

GBBA2:AF045481 2841 AF045481 Corynebacterium ammoniagenes, glucose permease (ptsG) gene.

complete cds.

Corynebacterium glutamicum Brevibacterium lactofermenlum Corynebacteriumn ammoniagenes 100,000 24-Nov.94 84,963 01-OCT-1993 53.558 29-Jul-98 -76r- Exemplification Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum C ATCC 13032 A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight Sat 30°C with vigorous shaking in BHI medium (Difco). The cells were harvested by 0 0 centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml Sbuffer-I of the original volume of the culture all indicated volumes have been 0 calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l MgSO, x 7HO, 10 ml/l KHPO, solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/1 M12 concentrate (10 g/l (NH,) 2 SO,, 1 g/l NaCI, 2 g/l MgSO, x 7H,O, 0.2 g/l CaCI,, 0.5 g/1 yeast extract (Difco), 10 ml/I trace-elements-mix (200 mg/l FeSO 4 x H 2 O, 10 mg/l ZnSO, x 7 HzO, 3 mg/l MnCI, x 4 HO, 30 mg/l HBO 3 20 mg/l CoCI, x 6 HO, 1 mg/l NiCI, x 6 H20, 3 mg/I Na 2 MoO, x 2 HO 2 500 mg/l complexing agent (EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/l biotin, 0.2 mg/I folic acid; mg/l p-amino benzoic acid, 20 mg/l riboflavin, 40 mg/l ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/1 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 ig/ml, the suspension is incubated for ca.18 h at 37°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 -200C 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 I0 min incubation at -20 0 C, the DNA was collected by centrifugation (13,000 rpm,

O

0 Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in I, TE-buffer. DNA prepared by this procedure could be used for all purposes, 4 including southern blotting or construction of genomic libraries.

EXAMPLE 2: Construction of genomic libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032 SUsing DNA prepared as described in Example 1, cosmid and plasmid o00 Slibraries were constructed according to known and well established methods (see c Sambrook, J. et al. (1989) "Molecular Cloning A Laboratory Manual", Cold

ID

0 10 Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) "Current Protocols ci 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. coil or other microorganisms Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain 78 r, the integrity of their genetic information. Typical mutator strains have mutations in the genes Sfor 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: C1 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.

O

00 Example 5: DNA Transfer Between Escherichia coli and Corynebacterium 0 glutamicum I0 Several Corynebacterium and Brevibacterium species contain endogenous 010 plasmids (as pHM1519 or pBL1) 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-

C

NI (1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for SC. 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 C, 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).

O Genes may be overexpressed in C. glutamicum strains using plasmids which 0 0 comprise pCG Patent No. 4,617,267) or fragments thereof, and optionally the O gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M. (1980) NO 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 ofmRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al.

C (1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer Sdesigned 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 C1 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 Sof mRNA for this gene. This information is evidence of the degree of transcription of the 00 mutant gene. Total cellular RNA can be prepared from Corynebacterium glutamicum by Sseveral methods, all well-known in the art, such as that described in Bormann, E.R. et al.

I (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 C',I 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 C which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH 4 CI or (NH,) 2

SO

4 NHOH, nitrates, urea, amino acids or O complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast 00 extract, meat extract and others.

SInorganic salt compounds which may be included in the media include the IDchloride-, 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 45°C. 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 -82r K is also possible to maintain a constant culture pH through the addition of NaOH or NHOH 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 Scompounds have high buffer capacities. If a fermentor is utilized for culturing the microorganisms, the pH can also be controlled using gaseous ammonia.

SThe incubation time is usually in a range from several hours to several days. This 00 time is selected in order to permit the maximal amount of product to accumulate in the Sbroth. The disclosed growth experiments can be carried out in a variety of vessels, such as I 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 600 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/1 polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/l 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 -83r, 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

C

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 0 3527300325); Bergmeyer, Bergmeyer, GraB1, eds. (1983-1986) Methods of SEnzymatic Analysis, 3 r d ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's SEncyclopedia 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) EMBO J. 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- "1 Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: D 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.

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

0 0 (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, SJ.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of O Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, 0 10 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 supernatant 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 (I1 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 Cchromatography 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 O impurities are retained by the resin while the sample is not. Such chromatography steps 0 0 may be repeated as necessary, using the same or different chromatography resins. One Sof ordinary skill in the art would be well-versed in the selection of appropriate I 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 -86r, 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST dprograms (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, "1 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 00 homologous to PTS protein molecules of the invention. To obtain gapped alignments Sfor comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., O (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.NA.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 of2, 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, e.g, 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 -87r" were 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 .I 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 O are aligned) was performed on these 500 hits. Each gene sequence of the invention was OO subsequently globally aligned to each of the top three FASTA hits, using the GAP Sprogram in the GCG software package (using standard parameters). In order to obtain Scorrect 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 ofDNA 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 D 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 C'1 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).

SThe 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 O 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 -89- N1 described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for Sexample, by the method of Shalon et al. (1996) Genome Research 6: 639-645).

The application of the sequences of the invention to DNA microarray CN1 technology, as described above, permits comparative analyses of different strains of C glutamicum or other Corynebacteria. For example, studies of inter-strain variations 0 based on individual transcript profiles and the identification of genes that are important 00 for specific and/or desired strain properties such as pathogenicity, productivity and Sstress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons \O 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 of electrophoretic 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: r" 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 "1 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 0 precursors 3S-methionine, "S-cysteine, 14C-labelled amino acids, 5 N-amino acids, 'N0 3 or NH4 or 3 C-labelled amino acids) in the medium of C. glutamicum S 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 et 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.

SEQUIVALENTS

c-I 3 Those of ordinary skill in the art will recognize, or will be able to ascertain Susing no more than routine experimentation, many equivalents to the specific c 5 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 00 0this specification are to be taken to specify the presence of stated features, c integers, steps or components or groups thereof, but do not preclude the 0 10 presence or addition of one or more other features, integers, steps, components c or groups thereof.

Claims (22)

1. An isolated Corynebacterium glutamicum nucleic acid molecule comprising a N nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID SSEQ ID NO:27 and SEQ ID NO:29, or a complement thereof. 00 CN 2. An isolated nucleic acid molecule which encodes a polypeptide comprising an 0 amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, Ci SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30, or a complement thereof.

3. An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30, 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:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29, or a complement thereof. An isolated nucleic acid molecule comprising a fragment of at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, or a complement thereof.

6. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1-5 and a nucleotide sequence encoding a heterologous polypeptide. N 7. A vector comprising the nucleic acid molecule of any one of claims 1-6. S8. The vector of claim 7, which is an expression vector.

9. A host cell transfected with the expression vector of claim 8. 0 The host cell of claim 9, wherein said cell is a microorganism. \O S11. The host cell of claim 10, wherein said cell belongs to the genus Corynebacterium C1 or Brevibacterium.

12. The host cell of claim 9, wherein the expression of said nucleic acid molecule results in the modulation in production of a fine chemical from said cell.

13. The host cell of claim 12, wherein said fine chemical is selected from the group consisting of: organic acids, proteinogenic amino acids, nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides, and enzymes.

14. A method of producing a polypeptide, the method comprising culturing the host cell of claim 9 in an appropriate culture medium to, thereby, produce the polypeptide. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID

16. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID SNO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID

17. An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleic acid sequence of O SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID 00 NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29. CN) \0 18. An isolated polypeptide comprising an amino acid sequence which is at least Sidentical to the entire amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID

19. An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence. The isolated polypeptide of any one of claims 15-19, further comprising heterologous amino acid sequences.

21. A method for producing a fine chemical, the method comprising culturing the cell of claim 9 such that the fine chemical is produced.

22. The method of claim 21, wherein said method further comprises the step of recovering the fine chemical from said culture.

23. The method of claim 21, wherein said cell belongs to the genus Corynebacterium or Brevibacterium. S24. The method of claim 21, 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, 0Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum, 00 0Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Cl Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum, \0 Sand those strains set forth in Table 3. The method of claim 21, wherein expression of the nucleic acid molecule from said vector results in modulation of production of said fine chemical.

26. The method of claim 21, wherein said finechemical is selected from the group consisting of: organic acids, proteinogenic amino acids, nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides, and enzymes.

27. The method of claim 21, wherein said fine chemical is an amino acid.

28. The method of claim 27, wherein said amino acid is drawn 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.

29. A method for producing a fine chemical, the method comprising culturing a cell whose genomic DNA has been altered by the inclusion of a nucleic acid molecule of any one of claims 1-6. INO 30. A method for diagnosing the presence or activity of Corynebacterium diphtheriae O in a subject, comprising detecting the presence of at least one of the nucleic acid molecules of any one of claims 1-5 or the polypeptide molecules of any one of claims S19, thereby diagnosing the presence or activity of Corynebacterium diphtheriae in the subject.

31. A host cell comprising a nucleic acid molecule selected from the group consisting 00 of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ 0 SID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, wherein the nucleic acid NO molecule is disrupted.

32. A host cell comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 or SEQ ID NO:29.

33. A host cell comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, wherein the regulatory region of the nucleic acid molecule is modified relative to the wild-type regulatory region of the molecule.

34. A polypeptide produced by the method of claim 14. A fine chemical produced by a method of any one of claims 21 to 29. DATED this 24 th day of February 2006 BASF AKTIENGESELLSCHAFT WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P20693AU01 seqI i st car r ect ed. t xt SEQUENCE LI STI NG <1 10> BASF Akt i engesel I schaft <120> COYNEBACTERI UM GLUTAM CUM GENES ENCODI NG PHOSPHONCL PYRUVATE: SUGAR PHOPHOTRANSFERASE SYSTEM PROTFEINS <130> BGI -122CPPC <140> POT! I BOO!00973 <141> 2000- 06- 27 <150> US 60/142, 691 <151> 1999- 07- 01 <150> US 60/ 150, 310 <151> 1999- 08-23 <150> DE 19942095.5 <151> 1999- 09- 03 <150> DE 19942097. 1 <151> 1999- 09- 03 <160> 36 <21 0> <211 <212> <213> <220> <221 <222> <223> 1 1 527 DNA Cor ynebact er i urn CIDS 101). (1504) RXS0031 gi ut ani cumn <400> 1 ct cat ggcat ct gcgccgt t cgcgttcttg ccagtgttgg ttggtttcac cgcaaccaag cgtttcggcg gcaatgagtt cctgggcgcc gcgtattggt gcg at g gt g Al a I'b t ValI ccg agc t tg gt g Pr o Ser Leu Val aac As n ggc tac gac gtg G y Tyr Asp Val gcc Al a 15 gcc acc at g gct Al a Thr I'bt Al a gcg ggc Al a 0 y gaa atg cca 0Gu I'bt Pro t gg t cc ct g tt t Tr p Ser Leu Phe ggt 0Gy t ta gat gt t gcc Leu Asp Val Al a caa gcc ggt a n Al a 0 y at t ct g gca I I e Leu Al a t ac cag ggc acc gt g ct t cct gt g Tyr a n a y Thr Val Leu Pro Val 45 ct g gt g gt t t ct Leu Val Val Ser t gg Tr p acg at c Thr I Ie gag aag ttc ctg a u Lys Phe Leu c ac Hi s aag cga ct c aag Lys Arg Leu Lys act gca gac tt c Thr Al a Asp Phe ct g Le u at c act cca gt g I Ie Thr Pro Val acg t tg ct g ct c Thr Leu Leu Leu ac c Thr gga t tc ct t aca a y Phe Leu Thr at c gcc at t ggc Se Al a IlIe a y cca Pr o gca atg cgc tgg Al a I'bt Ar g Tr p ggc gat gt g ct g a y Asp Val Leu gca cac Al a Hi s 1 00 Page 1 seql i st cor r ect ed. t xt ttc ggt ggt cca gtc ggc ggt ct a cag G y Leu G n ctt tat gat Leu Tyr Asp ttc Phe ttc Phe ttc Phe 150 gca Al a gct Al a ggt Gy gca Al a gcg Al a 230 gat Asp ttc Phe agc Ser aaa Lys at c SIle 310 gat Asp cca Pr o gca Al a ct g Leu 120 cca Pr o acg Thr ttc Phe ggt Gy aac Asn 200 at c l Ie ggc Gy gt c Val gca Al a gat Asp 280 gaa G u gca Al a at g Met aag Lys cca Pr o 360 t ac Tyr gag G u tct Ser ct g Leu 170 t cc Ser cgc Ar g ggc Gy gca Al a ttc Phe 250 at t l Ie gat Asp gaa G u ttg Leu gcc Al a 330 cag G n ggc Gy t ca Ser ct g Leu at g Met 155 gcg Al a gct Al a ct g Leu gct Al a ggt Gy 235 tt g Leu gct Al a gca Al a gca Al a acc Thr 315 agc Ser tta Leu cat Hi s cca Pr o gag G u 140 gct Al a aag Lys gtt Val cgc Ar g ttg Leu 220 ttc Phe gt g Val t at Tyr acc Thr ccc Pr o 300 ggt Gy gga Gy gtt Val gct Al a Pne at c SIle 125 ct g Leu aat Asn agt Ser ctt Leu tgg Tr p 205 at t SIle ttg Leu tgt Cys ggc Gy gct Al a 285 gca Al a gaa G u aag Lys tct Ser ttc Phe 365 u y 110 gt c Val ttt Phe at c SIle gaa G u ggt Gy 190 ccg Pr o gca Al a ggt Gy gca Al a ctt Leu 270 gct Al a gaa G u gct Al a ctt Leu ccg Pr o 350 gca Al a uy Pr o vai u y at c SIle aac Asn gcc Al a aag Lys 175 at t SIle ttc Phe ct c Leu gtt Val gtt Val 255 t ac Tyr cca Pr o ttt Phe at t l Ie ggc Gy 335 gt g Val gtt Val act Thr cag G n cag G n 160 ct c Leu acg Thr ttc Phe ttt Phe gtt Val 240 gtt Val ttg Leu gt g Val t ca Ser gca Al a 320 tcg Ser agt Ser cgc Ar g ggt Gy ggt Gy 145 ggt Gy aag Lys gag G u at c SIle aat Asn 225 tct Ser acc Thr gtt Val cct Pr o aac Asn 305 ct g Leu ggc gay gga Gy ct g Leu 130 gga Gy gcg Al a ggc Gy cct Pr o ggt Gy 210 at c SIle at t SIle ttc Phe cgc Ar g gca Al a 290 gat Asp agc Ser gtt Val aag Lys ggt ctg ctc G y Leu Leu 115 cac cag t cc Hi s G n Ser tcc ttc atc Ser Phe Ile gca tgt ttg Al a Cys Leu 165 ctt gca ggt Leu Al a G y 180 gcg atc ttc Al a i e Phe 195 at c ggt acc I e G y Thr aag gca gtt Lys Al a Val gat gct cca Asp Al a Pro 245 ttc atc gca Phe Ile Al a 260 cgc aac ggc Arg Asn G y 275 gga acg acc G y Thr Thr t cc acc at c Ser Thr Ile agc gtc agc Ser Val Ser 325 gcc at c gt c Al a I e Val 340 att gtg gtg Ile Val Val 355 gct gag gat Al a u Asp 451 499 547 595 643 691 739 787 835 883 931 979 1027 1075 1123 1171 1219 acc aag Thr Lys 370 Page 2 seqIi ggt t cc aat gt g gat at c t tg atg cac G31y Ser Asn Val As p I Ie Leu I'b t Hi s 375 380 ct c aac ggc acg cac t tt aac ccg ct g Leu As n G31y Thr Hi s Phe As n Pr o Leu 390 395 aaa gca ggg gag ct g ct g t gt gaa tt c Lys Al a G3 y G3 u Leu Leu Cys G3 u Phe 410 gca ggt t at gag gt a acc acg ccg at t Ala G31y Ty r (31u Val Thr Thr Pr o I Ie 425 430 acc gga cct gt a aac act t ac ggt t tg Thr G31y Pr o Val As n Thr Ty r G31y Leu 440 445 aac ct g ct c aac gt c gca aag aaa gaa Asn Leu Leu Asn Val Ala Lys Lys (31u 455 460 t aagt tgaaa cct tgagt gt t cg <210> 2 <211> 468 <212> PRT <213> Cor ynebact er i urn gi ut ani cumn <400> 2 I'b t Al a I'bt Val Phe Pr o Ser Leu Val 1 5 Thr I'b t Al a Al a G31y 31u I'b t Pr o I'bt Val Ala O31n Ala G31y Tyr O31n G31y Th r 40 Ser Tr p Ilie Leu Al a Thr Ilie O31u Lys 55 O31y Thr Al a Asp Phe Leu Ilie Thr Pr o 70 O31y Phe Leu Thr Phe I Ie Al a Ilie O31y Asp Val Leu Al a Hi s G31y Leu O31n G31y 100 105 Val G31y G31y Leu Leu Phe G31y Leu Val 115 120 G31y Leu Hi s O31n Ser Phe Pr o Pr o Ilie 130 135 G31y G31y Ser Phe Ilie Phe Al a Thr Al a 145 150 G31y Al a Al a Cys Leu Al a Val Phe Phe 1 65 Lys (3 y Leu Al a 03 y Al a Ser 03 y Val st cor r ect ed. t xt at t ggt t tc gac I I e 03 y Phe As p 385 aag aag cag ggc Lys Lys 03 n 03 y 400 gat at t gat gcc Asp Il e Asp Al a 415 gt t gt t t cg aat Val Val Ser Asn ggc gaa at t gaa G31y 31u Ilie O31u 450 gcg gt g cca gca Al a Val Pr o Al a 465 ac a Thr g at As p at t I Ie t ac Tyr 435 gcg Al a ac a Thr gt a ValI g aa (31u aag 420 aag gga G31y cca Pr o 1267 1315 1363 1411 1459 1 504 1 527 G31y Ser Le u Le u Le u Al a Tyr Ser Le u vb t 1 Al a Al a Tyr Le u Pr o Hi s Thr vb t As p Pr o O31u 140 Al a Lys5 ValI As p Ph e ValI Lys5 Le u Ar g Ph e I Ie 125 Le u As n Ser Le u Al a Le u ValI Le u Le u ValI Gy le As n Al a 1 li1e Page 3 seql i st cor r ect ed. t xt Su Pro Ala lie Phe Gy Val Asn Leu Arg Leu Arg 195 200 SIle Asn 225 Ser Thr Val Pr o Asn 305 Leu Gy Gy Thr Phe 385 G n Asp Ser SIle Pr o 465 SIle Lys Asp Phe Ar g 275 Gy Ser Ser Al a SIle 355 Al a Thr Asp SIle Tyr 435 Al a Thr Thr Val Pr o 245 Al a Gy Thr SIle Ser 325 Val Val Asp Asn Val 405 Al a Lys Al a Al a Al a 230 Asp Phe Ser Lys SIle 310 Asp Pr o Al a Gy Leu 390 Lys Al a Thr Asn Al a 215 Leu Lb t Gy I e Al a 295 G n Al a Thr Phe Ser 375 Asn Al a Gy Gy Leu 455 l Ie Gy Val Al a Asp 280 G u Al a Met Lys Pr o 360 Asn Gy Gy Tyr Pr o 440 Leu Gy Al a Mbet Al a 265 Pr o Al a Pr o Phe Gy 345 Ser Val Thr G u G u 425 Val Asn Gy Al a Phe 250 l le Asp G u Leu Al a 330 G n Gy Asp Hi s Leu 410 Val Asn Val Al a Gy 235 Leu Al a Al a Al a Thr 315 Ser Leu Hi s SIle Phe 395 Leu Thr Thr Al a Tr p 205 l le Leu Cys Gy Al a 285 Al a G u Lys Ser Phe 365 vbt Pr o G u Pr o Gy 445 Lys Pro Phe Phe Al a Gy Al a Leu 270 Al a G u Al a Leu Pr o 350 Al a Hi s Leu Phe SIle 430 Leu G u Leu Val Val 255 Tyr Pr o Phe SIe Gy 335 Val Val SIe Lys Asp 415 Val Gy Al a <210> 3 <211> 1109 <212> DNA <213> Corynebacter i um gl ut ai cum <220> <221> CDS <222> (1)..(1086) <223> FRXA00315 <400> 3 tat gat ttc ggc ggt cca gtc ggc ggt ctg ctc ttc ggt ctg gtc tac Page 4 Tyr Asp Phe G y 1 t ca Ser ct g Leu at g let gcg Al a gct Al a ct g Leu gct Al a ggt Sy ttg Leu 145 gct Al a gca Al a gca Al a acc Thr agc Ser 225 tta Leu cat Hi s at c SIle ct g Leu aat Asn agt Ser ctt Leu tgg Tr p at t l Ie 115 ttg Leu tgt Cys ggc G y gct Al a gca Al a 195 gaa G u aag Lys tct Ser ttc Phe Gy 5 at c SIle aac Asn gcc Al a aag Lys at t SIle ttc Phe ct c Leu gtt Val gtt Val t ac Tyr 165 cca Pr o ttt Phe at t SIle ggc Gy gt g Val 245 gtt Val act Thr cag G n cag G n ct c Leu 70 acg Thr tt c Phe ttt Phe gtt Val gtt Val 150 tt g Leu gt g Val t ca Ser gca Al a tcg Ser 230 agt Ser cgc Ar g ggt G y ggt Gy ggt Gy aag Lys gag G u at c SIle aat Asn tct Ser 135 acc Thr gtt Val cct Pr o aac Asn ct g Leu 215 ggc Gy gga Gy acc Thr ct g Leu gga Gy 40 gcg Al a ggc Gy cct Pr o ggt Gy at c SIle 120 at t SIle ttc Phe cgc Ar g gca Al a gat Asp 200 agc Ser gtt Val aag Lys aag Lys seql i st cor r ect ed. t xt Pro Val Gy y Leu Leu Phe y Leu Val Tyr cac Hi s t cc Ser gca Al a ctt Leu gcg Al a at c SIle 105 aag Lys gat Asp ttc Phe cgc Ar g gga G y 185 t cc Ser agc Ser gcc Al a at t SIle gct Al a 265 cag G n ttc Phe tgt Cys gca Al a at c I e 90 ggt Gy gca Al a gct Al a at c SIle aac Asn 170 acg Thr acc Thr gt c Val at c l Ie gt g Val 250 gag G u t cc Ser at c SIle ttg Leu ggt Gy 75 ttc Phe acc Thr gtt Val cca Pr o gca Al a 155 ggc G y acc Thr at c SIle agc Ser gt c Val 235 gt g Val gat Asp ttc Phe ttc Phe gca Al a gct Al a ggt Gy gca Al a gcg Al a gat Asp 140 ttc Phe agc Ser aaa Lys at c SIle gat Asp 220 cca Pr o gca Al a ggt Gy ccg Pr o gca Al a gt g Val t ca Ser gt g Val gct Al a ttg Leu 125 at g Met ggc G y at t SIle gcc Al a cag G n 205 gcc Al a acc Thr ttc Phe t cc Ser cca Pr o acg Thr ttc Phe ggt G y aac Asn at c l Ie 110 ggc G y gt c Val gca Al a gat Asp gaa G u 190 gca Al a at g Met aag Lys cca Pr o aat Asn 270 at t SIle gca Al a ttc Phe gt c Val ctt Leu ggt G y gct Al a at g Met gcg Al a cca Pr o 175 gca Al a cct Pr o ttt Phe ggg G y tct Ser 255 gt g Val gag G u tct Ser ctg Leu t cc Ser cgc Ar g ggc G y gca Al a ttc Phe att SIe 160 gat Asp gaa G u ttg Leu gcc Al a cag G n 240 ggc G y gat Asp atc ttg atg cac att ggt ttc gac aca gta aac ctc aac ggc acg cac Page I Ie Leu I'b t His Ilie y Phe Asp T 275 280 ttt aac ccg ctg aag aag cag ggc g Phe Asn Pr o Leu Lys Lys O31n Gy1 y 290 295 ct g t gt gaa t tc gat at t gat gcc a Leu Cys 03 u Phe Asp Il e Asp Al a 1 305 310 acc acg ccg at t gt t gt t t cg aat t Thr Thr Pr o Ilie Val Val Ser Asn T 325 act t ac ggt t tg ggc gaa at t gaa g Thr Tyr 03 y Leu 03 y 03 u Il e 03 u P 3403 gca aag aaa gaa gcg gt g cca gca a Al a Lys Lys (31u Al a Val Pr o Al a T 355 360 t cg <210> 4 <211> 362 <21 2> PRT <21 3> Cor ynebact er i urn gi ut anm cumn lIi st car r ect ed. t xt hr Val Asn Leu Asn Gy Thr His 285 iat gaa gt c aaa gca ggg gag ct g osp (31u Val Lys Al a G31y 31u Leu 300 .tt aag gct gca ggt t at gag gt a l e Lys Al a Al a 03 y Tyr 03 u Val 315 320 ac aag aaa acc gga cct gta aac yr Lys Lys Thr G31y Pr o Val Asn 330 335 icg gga gcc aac ct g ct C aac gt c Il a 03 y Al a Asn Leu Leu Asn Val 45 350 ca cca t aagt t gaaa cct t gagt gt hr Pr o 912 960 1 008 1 056 1106 1109 <400> 4 Tyr Asp Ser Pr o Leu 03 u [vb t Al a Al a Lys Al a Val Leu Arg Al a Leu 03 y Phe 130 Leu Val 145 Al a Ty r Phe G31y G31y Pr o Val li1e Val I Ie Th r G31y Leu Phe As n 03 n 03 y Asn I I e Al a 03 n 03 y 55 Ser 03 u Lys Leu Lys 70 Leu 03 y I I e Thr 03 u Tr p Pr o Phe Phe I Ie 1 00 I I e Al a Leu Phe As n 115 Leu 03 y Val Val Ser 135 Cys Al a Val Val Thr 150 03 y Leu Ty r Leu Val 1 65 o31yo(1y Leu Hi s 25 03 y Se r 40 Al a Al a 03 y Leu Pr o Al a G31y I Ie 1 05 1I e Lys 120 1 1 e As p Phe Phe Ar g Ar g Leu Leu Phe 10 03 n Ser Phe Phe Il e Phe Cys Leu Al a Al a 03 y Al a Il e Phe 03 y 90 03 y Thr Al a Al a Val Al a Al a Pr o Asp 140 Il e Al a Phe 1 Asn G31y Ser 1 70 Thr Thr Lys o31y Pr a Al a ValI Ser ValI Al a Le u 125 vb t oy le Al a Le u Pr a Thr Ph e o31y As n I Ie 110 o31y ValI Al a As p O31u 1 ValI lie Al a Ph e ValI Le u o31y Al a vb t Al a Pr a 1 Al a Ala Thr Ala Ala Pro Val Pro Ala 1 80 o31y 1 85 Page 6 Al a Thr Ser 225 Leu Hi s SIle Phe Leu 305 Thr Thr Al a Al a 195 G u Lys Ser Phe bet 275 Pr o G u Pr o Gy Lys 355 Phe SIle Gy Val 245 Val SI e Lys Asp Val 325 Gy Al a Ser Al a Ser 230 Ser Ar g Gy Lys SIle 310 Val G u Val Asn Leu 215 Gy Gy Thr Phe G n 295 Asp Ser SIle Pr o seql i st cor r ect ed. t xt Asp Ser Thr lie lie n 200 205 Ser Ser Val Ser Asp Al a 220 Val Ala lie Val Pro Thr 235 Lys lie Val Val Ala Phe 250 Lys Al a u Asp G y Ser 265 Asp Thr Val Asn Leu Asn 280 285 Sy Asp G u Val Lys Ala 300 Al a lie Lys Al a Al a G y 315 Asn Tyr Lys Lys Thr y 330 Su Ala y Ala Asn Leu 345 Al a Thr Pro 360 Al a Met Lys Pr o Asn 270 Gy Gy Tyr Pr o Leu 350 Pr o Phe Gy Ser 255 Val Thr G u G u Val 335 Asn <210> <211> 372 <212> DNA <213> Corynebacter i um gl ut am cum <220> <221> CDS <222> (101)..(349) <223> RXA01503 <400> gtatcctcaa aggcct t cta gctgt t gcag ctgcagcgca cgacctatca aattctttat gctgcaggcg atgccttttc at t l Ie t ca Ser acc Thr caa G n cag gcg Al a gca Al a ttg Leu gct Al a gcg act Thr gct Al a gac Asp aat Asn cat gcg Al a ggt Gy ggt Gy gac Asp gt a gct Al a gca Al a gaa G u gt c Val gcg aaa Lys ct g Leu cag G n ttc Phe ttg ttc ggt Phe G y 15 cac aca Hi s Thr tcg atg Ser Vbet ctg ggc Leu G y atg aag Page 7 ctcggtggat acgacatcca atg ttc ttg gca gtc IVet Phe Leu Ala Val 1 gcc aat gtc ttt aca Al a Asn Val Phe Thr cag ctt cag gca gta G n Leu G n Al a Val act ctg gtg gct ttc Thr Leu Val Ala Phe att cca gtg gtg ctg Ile Pro Val Val Leu ttg tcg cga seql i st cor r ect ed. t xt G n Leu Ala Leu His Val Ala Ser Leu IVet Lys Leu Ser Arg 75 taagaggagg ggcgtgtcgg tct <210> 6 <211> 83 <212> PRT <213> Cor ynebact eri um gl ut am cum <400> 6 IVet Phe Leu Ala Val Ile Leu Ala lIe Thr Al a Ala Arg Lys Phe y 1 5 10 Ala Asn Val Phe Thr Ser Val Ala Leu Ala G y Ala Leu Leu His Thr 25 G n Leu G n Ala Val Thr Val Leu Val Asp Gy G u Leu G n Ser Met 40 Thr Leu Val Ala Phe G n Lys Ala y Asn Asp Val Thr Phe Leu y 55 Ile Pro Val Val Leu G n Leu Ala Leu His Val Ala Ser Leu IVet Lys 70 75 Leu Ser Arg <210> 7 <211> 2187 <212> DNA <213> Cor ynebact eri um gl ut am cum <220> <221> CDS <222> (101)..(2164) <223> RXN01299 <400> 7 cgactgcggc gtctcttcct ggcactacca ttcctcgtcc gtgcaacggt cacccaagtc aaaggattga aagaatcagc aat Asn gat Asp t cc Ser t cc Ser gaa Gu tcg Ser at c SIle gcc Al a acc Thr gt a Val gt c Val aac Asn gcc Al a gtt Val gt c Val ct g Leu gcc Al a gcc Al a ggt Gy acc Thr gat Asp act Thr aaa Lys 45 caa G n ttg Leu gt c Val gtt Val gac Asp gtt Val ggc Gy gat ttc Asp Phe 15 att ttc I e Phe gcg ctg Al a Leu gct at c Ala I e ttt gct Phe Al a 80 Page 8 tgaccaactc gccacagctg atg aat agc gta aat 115 IVet Asn Ser Val Asn 1 ggc gac tcc acc acg 163 G y Asp Ser Thr Thr gac gct ggc cga gct 211 Asp Ala Gy Arg Ala gat cgt gaa gca aag 259 Asp Arg G u Ala Lys ccc cac tgc cgt tcc 307 Pro His Cys Arg Ser cgc ctg agc aag ggt 355 Arg Leu Ser Lys y gtg gac ttc agc Val Asp Phe Ser gca Al a ctt Leu gcc Al a cca Pr o 150 gtt Val at c SIle t cc Ser gag G u gaa G u 230 cgc Ar g gcg Al a aag Lys gct Al a gca Al a 310 ggc Gy aac Asn cct Pr o cgc Ar g 120 acc Thr cca Pr o gag G u gca Al a acg Thr 200 cag G n gcc Al a cgt Ar g aat Asn cca Pr o 280 acc Thr at g Met ct g Leu tgg Tr p gga Gy ggc Gy ttg Leu cag G n acc Thr ggg Sy 170 cca Pr o aac Asn tct Ser gcc Al a gct Al a 250 cca Pr o gcc Al a ggc Sy ggc Sy gct Al a 330 gca Al a ggc Gy aag Lys at c SIle 140 gag G u gcg Al a ggt Gy gaa G u gct Al a 220 at c l Ie aag Lys aag Lys aag Lys aag Lys 300 t cc Ser ggc G y gcc Al a aaa Lys aag Lys 125 gt c Val cca Pr o tcg Ser at c SIle ggc Sy 205 gt c Val ttc Phe cca Pr o at g Met gtt Val 285 ct c Leu t ac Tyr ttc Phe acc Thr gag G u 110 gat Asp gac Asp gct Al a aca Thr gca Al a 190 cgc Ar g acc Thr gcc Al a gt c Val at c SIle 270 t cc Ser ggc Gy at g let gca Al a cag G n 350 seql i st cor r ect ed. t cct gat ggc gat gcc aac ttg Pro Asp y Asp Ala Asn Leu gtg ttc ctc att Val Phe Leu Ile ct g Leu at c SIle gt c Val gct Al a 160 gtt Val acc Thr gat Asp gt c Val gac Asp 240 gaa G u gag G u t cc Ser ggc Gy cca Pr o 320 ggt Gy tct Ser aag Lys aag Lys gat Asp 145 ccg Pr o act Thr t ac Tyr gt g Val gat Asp 225 gt g Val t cc Ser gcc Al a ggt Gy aag Lys 305 ttc Phe gga Gy ct g Leu at c SIle gct Al a 130 gcc Al a gct Al a cgt Ar g at g Met gaa G u 210 ccg Pr o gga Gy ggc Gy at c l Ie gt c Val 290 cgc Ar g gt a Val t ac Tyr acc Thr ct g Leu 115 ct g Leu gt g Val gcg Al a at c SIle gct Al a 195 ct c Leu aag Lys gtt Val gt c Val gca Al a 275 gcg Al a at c SIle gct Al a gac Asp aac Asn 355 100 t cc Ser cag G n ct c Leu gcg Al a gt g Val 180 gcg Al a gtt Val at c SIle aaa Lys aag Lys 260 gcc Al a gca Al a cag G n gcc Al a at g Met 340 ct g Leu aag Lys gaa G u aac Asn gcg Al a 165 gca Al a gat Asp gt g Val at c SIe gac Asp 245 cgc Ar g t cc Ser tct Ser cag G n ggc G y 325 gcg Al a cca Pr o 403 451 499 547 595 643 691 739 787 835 883 931 979 1027 1075 1123 1171 Page 9 Sc C ggc aac acc gtc gat gtt gac Sy Asn Thr Val Asp Val Asp 360 gg eql i st cor r ect ed. t xt gtg gcc atg acc ttc Val Ala lMt Thr Phe gag G u ggc Gy gcc Al a 390 ctt Leu t cc Ser ttg Leu gt g Val gt g Val 470 t cc Ser t cc Ser ct c Leu ct g Leu gca Al a 550 cgc Ar g tgg Tr p gca Al a ct g Leu ggc Gy gga Gy acc Thr ggt Gy 440 cag G n gtt Val at g Met at c SIle gga Gy 520 acc Thr ggc Gy aag Lys ctt Leu gac Asp 600 t ac Tyr at c SIle cca Pr o 410 ggc Gy at t SIle ct g Leu ct c Leu ggt Gy 490 ct g Leu gt a Val gac Asp gt c Val ttc Phe 570 ct g Leu ttc Phe tt c Phe gt g Val 395 ggc Gy gct Al a gcc Al a at g bet gt c Val 475 tt g Leu ggt Gy aac Asn caa G n cca Pr o 555 acc Thr gca Al a cgt Ar g ggc Gy 380 gca Al a at c SIle ggc Gy ct g Leu cct Pr o 460 at g Met cag G n at c SIle aag Lys gct Al a 540 cca Pr o cca Pr o ttc Phe gt g Val u y 365 gca Al a gcc Al a gcg Al a ttc Phe tgg Tr p 445 gt g Val t ac Tyr gac Asp at c SIle gca Al a 525 t cc Ser at c SIle gca Al a gt c Val at c SIle 605 gt c Val ct g Leu ccg Pr o at t SIle 430 at t SIle gt c Val ct c Leu tgg Tr p ttg Leu 510 gcc Al a at g Met gcg Al a gag G u t cc Ser 590 cca Pr o ct g Leu tct Ser ggc Gy 415 ggt Gy ggc Gy at c SIle ct g Leu ct a Leu 495 ggc Gy t ac Tyr gaa G u ttg Leu caa G n 575 gaa G u gca Al a ttc Phe ggc Gy 400 ttc Phe ggt Gy t cc Ser at c SIle ct g Leu 480 tcg Ser ct c Leu ct c Leu at c SIle t cc Ser 560 gaa G u ggt Gy at g Met gt c Val gcc Al a 385 t ac Tyr gt c Val ct g Leu tgg Tr p ccg Pr o 465 ggt Gy t ca Ser at g Met tt t ttt Phe at g Met 545 at t SIle aac Asn gcc Al a at g Met ggc G5y 625 370 acc ggc Thr G y acc gca Thr Al a ggt ggc Gy Gy gtt acc Val Thr 435 aag gtg Lys Val 450 ct a ct t Leu Leu cgc cca Arg Pro atg t cc let Ser at g t gt Met Cys 515 ggt acc G y Thr 530 gcc gcg Al a Al a gct acc Al a Thr ggc aag G y Lys atc cca lie Pro 595 gct ggc Al a G y 610 tct cgg Ser Arg cgt Ar g caa G n t ac Tyr gcc Al a 420 ggt Gy cca Pr o acc Thr ct c Leu gga Gy 500 ttc Phe gca Al a at c l Ie ct g Leu t ct Ser 580 ttc Phe ggt Gy gct Al a t ca Ser gca Al a gca Al a 405 at c SIe at c SIe cgc Ar g t ca Ser gca Al a 485 agc Ser gac Asp ggc Gy at g Met ct g Leu 565 t cc Ser gcc Al a gca Al a cca Pr o 1219 1267 1315 1363 1411 1459 1507 1555 1603 1651 1699 1747 1795 1843 1891 1939 1987 acc act ggt gca atc tcc atg gca ctg ggc Thr Thr y Ala lie Ser IVet Ala Leu y 615 620 Page cac ggc ggt atc ttc gtg gtc tgg g His G y y I I e Phe Val Val Trp A 630 635 ctc atc gca ctt gca gca ggc acc a Leu lie Ala Leu Ala Ala y Thr I 650 gca ctg aag cag ttc tgg cca aac a Ala Leu Lys G n Phe Trp Pro Asn L 665 6 aag caa gaa gca caa caa gca gct g Lys n G u Al a n G n Al a Ala 680 685 gt c <210> 8 <211> 688 <212> PRT <213> Corynebacter i um gl ut ai cum 1 i st cor r ect ed. t xt ica atc gaa cca tgg tgg ggc tgg I a lie G u Pro Trp Trp y Trp 640 645 Itc gtg tcc acc atc gtt gtc atc le Val Ser Thr Ile Val Val Ile 655 660 .ag gcc gtc gct gca gaa gtc gcg ys Ala Val Ala Ala G u Val Ala 70 675 ita aac gca taatcggacc ttgacccgat (al Asn Ala 2035 2083 2131 2184 2187 <400> 8 Ivbt Asn Ser 1 G y Asp Ser Asp Al a G y Asp Arg u Pro His Cys Arg Leu Ser Leu Val Phe Lys Ile Leu 115 Lys Ala Leu 130 Asp Al a Val 145 Pr o Al a Al a Thr Arg Ile Tyr IVet Al a 195 Val G u Leu 210 Val Asn Asn Ser 5 Thr Thr Asp Val Arg Ala Ser Ser Al a Lys Ser G y 55 Arg Ser G u Ala 70 Lys y Val Asp Leu Ile Al a Al a 100 Ser Lys Leu Al a G n G u Al a Thr 135 Leu Asn Pro Ala 150 Ala Ala Val Ala 165 Val Ala I e Thr 180 Ala Asp Ser Leu Val Val G u Thr 215 Ser Leu Val 10 Ile Asn Asn 25 Al a Asp Al a 40 Thr G y Val Val Ser Val Phe Ser G y 90 Pro Al a G y 105 Arg Ser Leu 120 Thr G u G n Pro Lys Thr Su Ser G y 170 Al a Cys Pr o 185 Thr G n Asn 200 Sn G y Ser Ar g Leu Leu Pr o Pr o 75 Pr o Sy Val G u Thr 155 Al a Thr Al a Ser Leu Al a Al a Sy Thr Asp Sy Lys SIle 140 G u Al a Sy G u Al a 220 Asp Thr Lys G n Leu Sy Lys Lys 125 Val Pr o Ser SIle Sy 205 Val Val Val Asp Val Sy Asp G u 110 Asp Asp Al a Thr Al a 190 Ar g Thr Asp I e Al a Al a Phe Al a Hi s Phe Val Al a Ser 175 Hi s Asp Pr o Phe Phe Leu SIe Al a Asn Leu SIe Val Al a 160 Val Thr Asp Val Asp Pro Lys Ile Ile Gu Ala Ala Asp Ala Val Page 11 Ile Phe Ala Thr Asp seqi i st car r ect ed. t xt 225 ValI Ser A a G31y Ly s 305 Ph e G31y Le u Thr A a 385 Tyr ValI Le u Tr p Pr o 465 G31y Ser vb t Ph e vb t 545 li1e 235 (31y Lys Pro Val ValI ValI A a 275 A a I Ie A a As p As n 355 O31u G31y A a G31y Thr 435 ValI Le u Pr o Ser cys 515 Thr A a Thr As p 245 Ar g Ser Ser O31n G31y 325 A a Pr o Ser A a A a 405 li1e I Ie Ar g Ser A a 485 Ser As p G31y vb t Le u 565 Ar g O31u Ar g Ph e A a Ly s A a A a 310 G31y As n G31y G31y A a 390 Le u Ser Le u ValI ValI 470 Ser Ser Le u Le u A a 550 Ar g I Ie As n (31u 295 ValI Le u G31y As n Ph e 375 vb t A a ValI A a ValI 455 ValI I Ie A a G31y Ser 535 A a Ly s (31u 265 As n Thr Thr Le u O31n 345 ValI Le u Ph e Ar g I Ie 425 Le u Ser G31y Thr Le u 505 Pr o G31y vb t Le u G31y 585 A a 250 Pr o A a G31y G31y A a 330 A a As p Tyr I Ie Pr a 410 G31y I Ie Le u Le u G31y 490 Le u ValI As p ValI Ph e 570 A a Ar g O31u ValI 315 Le u I Ie ValI Ph e ValI 395 G31y A a A a vb t ValI 475 Le u G31y As n O31n Pr a 555 Thr ValI 285 Le u Tyr Ph e Thr G31y 365 A a A a A a Ph e Tr p 445 ValI Tyr As p I Ie A a 525 Ser I Ie A a I Ie 270 Ser G31y vb t A a O31n 350 ValI ValI Le u Pr a I Ie 430 li1e ValI Le u Tr p Le u 510 A a vb t A a (31u 240 li1e O31u 255 As p (1u O3 y Ser Trp O31y Val Pr o 320 Phe 03 y 335 Phe Ser A a [Vb t Leu Phe Ser 03 y 400 O3 y Phe 415 O31y (1y O3 y Ser I Ie I Ie Leu Leu 480 Leu Ser 495 O3 y Leu Tyr Leu O31u I Ie Leu Ser 560 O31n (1u 575 Asn O31y Lys Ser Ser Tr p Leu Leu 580 Leu Ala Phe Val Ser O31u O31y 590 Page 12 Al a vb t G31y 625 Pr o Thr Al a Pr o 595 G31y Ar g Tr p ValI (31u 675 Al a Al a Pr o Tr p 645 li1e Al a Al a Thr Hi s 630 Le u Al a Ly s Al a Thr 615 Oy le Le u O31n seqI i st car r ect ed. t xt Asp Pro Phe Arg Val I Ie 600 605 03 y Al a I I e Ser I'b t Al a 620 O31y I Ie Phe Val Val Tr p 635 Al a Leu Al a Al a 03 y Thr 650 Lys O31n Phe Tr p Pr o As n 665 03 u Al a 03 n 03 n Al a Al a 680 685 Pr o Le u Al a I Ie Ly s 670 ValI Al a G31y I Ie ValI 655 Al a As n <210O> 9 <21 1> 464 <212> DNA <213> Cor ynebact er i urn gi ut ari cum <220> <221 CIDS <222> (441) <223> FRXA01299 <400> 9 atg gaa gcg tt g Al a Leu gag caa O31u (1n t cc gaa Ser 03 u cca gca Pr o Al a ct g ggc Leu 03 y gca at c Al a I I e at c gt g I e Val at c I Ie t cc Ser g aa O31u ggt G31y at g vb t gt c ValI g aa O31u t cc Ser 115 gcc Al a gct Al a ggC G31y at c I Ie gct Al a t ct Ser t gg Tr p at c I Ie gcg atc atg gca gct ggc atg gtc cca cca atc Ala Ilie I'bt Ala Ala Gy I'bt Val Pro Pro Ilie 10 ct g Le u t ct Ser tt c Ph e 55 ggt G31y gct Al a ggC G31y gt c ValI ct g Le u t cc Ser 40 gcc Al a gca Al a cca Pr a t gg Tr p at c I Ie 120 cgc Ar g t gg Tr p gca Al a ac c Thr c ac Hi s ct c Le u 1 05 gca Al a aag Ly s ctt Le u g ac As p ggt G31y 75 ggt G31y gca Al a aag Ly s ct g Le u ggC G31y cca Pr a 60 gca Al a at c I Ie ctt Le u C ag O31n tt c Ph e ct g Le u 45 tt c Ph e at c I Ie tt c Ph e gca Al a tt c Ph e 125 ac c Thr 30 gca Al a cgt Ar g t cc Ser gt g ValI gca Al a 110 t gg Tr p cca gca Pr o Al a t tc gt c Phe Val gt g at c Val I Ie at g gca [vb t Al a gt c t gg Val Tr p ggc acc 03 y Thr cca aac Pr o As n aag gcc gt c gct gca gaa gt c gcg aag caa gaa gca caa caa gca gct Lys AlaVal AiaAiaOu Val Aa LysOnOu Aa OnOn AlaAla Page 13 seql i st cor r ect ed. t xt gta aac gca taatcggacc ttgacccgat gtc Val Asn Ala 145 <210> <211> 147 <212> PRT <213> Cor ynebact eri um gl ut arri cum <400> Vet u Ile Vet Ala Ala lle Iet Ala Ala Oy Iet Va 1 5 10 Ala Leu Ser lie Ala Thr Leu Leu Arg Lys Lys Leu Ph 25 G u n G u Asn y Lys Ser Ser Trp Leu Leu y Le 40 4 Ser G u y Ala lie Pro Phe Alaa Aa Ala Asp Pro Ph 55 Pro Ala Met Met Ala y y Ala Thr Thr y Ala I 70 75 Leu G y Val G y Ser Arg Ala Pro His y G y I I e Ph 90 Ala lie u Pro Trp Trp y Trp Leu lie Ala Leu Al 100 105 lie Val Ser Thr lie Val Val Ile Ala Leu Lys G n Ph 115 120 12 Lys Ala Val Ala Ala G u Val Ala Lys n G u Ala G 130 135 140 Val Asn Ala 145 <210> 11 <211> 580 <212> DNA <213> Cor ynebact eri um gl ut arri cum <220> <221> CDS <222> (101)..(580) <223> FRXA01883 <400> 11 cgactgcggc gtctct t cct ggcactacca ttcctcgtcc tgacca gtgcaacggt cacccaagtc aaaggattga aagaatcagc atg aa e u e e e a e n Pr o Thr Al a Ar g Ser Val Al a 110 Tr p G n Pr o Pr o Phe Val Met Val Sy Pr o Al a SIe Al a Val SIe Al a Tr p Thr Asn Al a actc gccacagctg t agc gta aat aat tcc tcg ctt gtc cgg ctg gat gtc gat ttc Asn Ser Ser Leu Val Arg Leu Asp Val Asp Phe 15 gat gtc atc aac aac ctt gcc act gtt att ttc Asp Val lie Asn Asn Leu Ala Thr Val lie Phe 30 Page 14 IVet Asn Ser Val Asn 1 ggc gac tcc acc acg G y Asp Ser Thr Thr gac gct ggc cga gct Asp Ala y Arg Ala seql i st cor tcc tcc gcc gac gcc ctt gcc aaa gac gcg Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala 45 t cc ggc acc ggc gtt cct ggt caa gtt gct Ser G y Thr G y Val Pro G y G n Val Al a 60 gaa gcc gta tct gtc cct acc ttg ggc ttt G u Ala Val Ser Val Pro Thr Leu y Phe 75 gtg gac ttc agc gga cct gat ggc gat gcc Val Asp Phe Ser Gy Pro Asp y Asp Al a 95 gca gca cct gct ggc ggc ggc aaa gag cac Ala Ala Pro Ala G y G y G y Lys G u Hi s 105 110 ctt gct cgc tcc ttg gtg aag aag gat ttc Leu Ala Arg Ser Leu Val Lys Lys Asp Phe 120 125 gcc acc acc gag cag gaa atc gtc gac gtt Ala Thr Thr G u n G u Ile Val Asp Val 135 140 cca gca cca aaa aac cac cga gcc agc tgc Pro Ala Pro Lys Asn His Arg Ala Ser Cys 150 155 <210> 12 <211> 160 <212> PRT <213> Corynebacter i um gl ut ai cum <400> 12 IVet Asn Ser Val Asn Asn Ser Ser Leu Val 1 5 10 Sy Asp Ser Thr Thr Asp Val lie Asn Asn Asp Ala y Arg Ala Ser Ser Ala Asp Al a 40 Asp Arg G u Ala Lys Ser y Thr y Val 55 Pro His Cys Arg Ser G u Ala Val Ser Val 70 Arg Leu Ser Lys y Val Asp Phe Ser y 90 Leu Val Phe Leu lie Al a Ala Pro Ala y 100 105 Lys lie Leu Ser Lys Leu Ala Arg Ser Leu 115 120 Lys Ala Leu n G u Al a Thr Thr G u G n 130 135 Asp Al a Val Leu Asn Pro Ala Pro Lys Asn 145 150 Pag r ect ed. txt ctg gat cgt Leu Asp Arg atc ccc cac lie Pro His gct cgc ct g Al a Ar g Leu aac ttg gtg Asn Leu Val ct g aag at c Leu Lys II e at c aag gct I e Lys Al a 130 gtc gat gcc Val Asp Al a 145 agc Ser 160 gaa G u tgc Cys agc Ser ttc Phe ct g Leu 115 ct g Leu gt g Val gca Al a cgt Ar g aag Lys ct c Leu 100 t cc Ser cag G n ct c Leu Ar g Leu Leu Pr o Pr o Pr o Sy Val G u Hi s 155 e Leu Al a Al a Sy Thr Asp Sy Lys SIle 140 Ar g Asp Thr Lys G n Leu Sy Lys Lys 125 Val Al a Asp I e Al a Al a Phe Al a Hi s Phe Val Cys seql i st cor r ect ed. t xt <210> <211> <212> <213> <220> <221> <222> <223> 13 631 DNA Cor ynebact eri um gl ut am cum CDS (631) FRXA01889 <400> 13 accgagccag ctgcagctcc ggctgcggcg gccggttgtt aagagtgggg cggcgtcgac aagcgt t act acc t ac at c Thr Tyr ?bet cgtatc gt g Val 1 gca atc acc gca tgc cca acc ggt atc gca cac Ala IIe Thr Ala Cys Pro Thr G y IIe Ala His 5 112 160 gct gcg gat t cc Al a Al a Asp Ser acg caa aac gcg Thr G n Asn Al a ggc cgc gat G y Arg Asp gat gtg Asp Val gaa ctc gt t G u Leu Val gtg gag Val G u act cag ggc tct Thr G n y Ser gct gtc acc cca Ala Val Thr Pro gtc gat ccg aag atc atc gaa gct gcc gac gcc gtc atc ttc gcc acc Val Asp Pro Lys Ile Ile u Ala Ala Asp Ala Val lie Phe Ala Thr 50 55 gac gtg gga gtt Asp Val y Val aaa Lys gac cgc gag cgt Asp Arg G u Arg gct ggc aag cca Ala y Lys Pro gtc att Val lie gaa tcc ggc G u Ser G y gag gcc atc G u Ala le aag cgc gcg atc Lys Arg Ala lle aat Asn gag cca gcc aag Su Pro Ala Lys at g atc gac IVet Ile Asp gtt tcc ggt Val Ser G y gca gcc tcc aag Ala Ala Ser Lys cca aac gcc cgc Pro Asn Ala Arg aag Lys 105 tcc ggt Ser G y 110 gtc gcg gca tct Val Ala Ala Ser gct Al a 115 gaa acc acc ggc G u Thr Thr G y aag ctc ggc tgg Lys Leu y Trp ggc Sy 125 aag cgc atc cag Lys Arg lie n gca gtc atg acc Ala Val Met Thr ggc Sy 135 gtg tcc tac atg Val Ser Tyr bet cca ttc gta gct Pro Phe Val Ala gcc Al a 145 ggc ggc ctc ctg G y G y Leu Leu gct ctc ggc ttc Al a Leu G y Phe gca ttc Al a Phe 155 ggt gga tac G y G y Tyr tct ctg acc Ser Leu Thr 175 atg gcg aac ggc IVet Al a Asn G y tgg Tr p 165 caa gca atc gcc G n Ala lle Ala acc cag ttc Thr G n Phe 170 aac ctg cca ggc Asn Leu Pro G y acc gtc gat gtt Thr Val Asp Val gac Asp 185 <210> 14 Page 16 seql i st cor r ect ed. t xt <211> 185 <212> PRT <213> Corynebact eri um gl ut am cum <400> 14 Val Ala IIe Thr Ala Cys 1 Al a Val SIle Lys Lys Al a Al a G n Al a 145 eut Leu Ser G u G u Ar g Al a Lys Al a 115 Al a Gy Asn Gy 5 Thr G n Al a Ar g Asn Pr o Thr bet Leu Tr p 165 Thr Pr o Asn Ser Al a Al a Pr o Al a Gy Gy 135 Al a Al a Asp Thr Al a Ser 40 Val Gy Al a Ar g G u 120 Val Leu l Ie Val Gy G u Al a SIle Lys Lys Lys 105 Lys Ser Gy Al a Asp 185 Al a Ar g Thr Al a Val I le Ser Gy et Al a 155 G n Thr Asp Val Asp G u G u Ser Gy 125 Pr o Gy Ser ebt G u Pr o Gy Gy I e Val Ar g Val Tyr Thr 175 <210> <211> 416 <212> DNA <213> Corynebacter i um gl ut am cum <220> <221> CDS <222> <223> RXA00951 <400> atc caa gca atc tta gag aag gca gca Ile G n Ala l e Leu u Lys Ala Al a 1 5 cct gct gtg gct cct gct gta aca ccc Pro Ala Val Ala Pro Ala Val Thr Pro 25 gtc caa tcc aaa acc cac gac aag atc Val G n Ser Lys Thr His Asp Lys Ile 40 ttg ggt acc tcc ctc ttc ctc aaa aac Leu G y Thr Ser Leu Phe Leu Lys Asn 55 gcg ccg gcg aag cag aag gct Ala Pro Ala Lys n Lys Ala 10 act gac gct cct gca gcc tca Thr Asp Ala Pro Ala Ala Ser ctc acc gtc tgt ggc aac ggc Leu Thr Val Cys Gy Asn y acc ctt gag caa gtt ttc gac Thr Leu G u G n Val Phe Asp Page 17 ^0 seql i st cor r ect ed. t xt acc tgg ggt tgg ggt cca tac atg acg gtg gag gca acc gac act atc 240 SThr Trp G y Trp G y Pro Tyr Vet Thr Val G u Ala Thr Asp Thr Ile 70 75 tcc gcc aag ggc aaa gcc aag gaa gct gat ctc atc atg acc tct ggt 288 L Ser Ala Lys G y Lys Al a Lys u Ala Asp Leu lie et Thr Ser G y 90 gaa atc gcc cgc acg ttg ggt gat gtt gga atc ccg gtt cac gtg atc 336 Su Ile Ala Arg Thr Leu y Asp Val y Ile Pro Val His Val lie 100 105 110 0 aat gac ttc acg agc acc gat gaa atc gat gct gcg ctt cgt gaa cgc 384 00 Asn Asp Phe Thr Ser Thr Asp G u lie Asp Ala Ala Leu Arg G u Arg 115 120 125 C1 tac gac atc taactacttt aaaaggacga aaa 416 sC) Tyr Asp Ile S 130 <210> 16 <211> 131 <212> PRT <213> Corynebacter i um gl ut ari cum <400> 16 lie n Ala lie Leu u Lys Ala Ala Ala Pro Ala Lys n Lys Ala 1 5 10 Pro Ala Val Ala Pro Ala Val Thr Pro Thr Asp Ala Pro Ala Ala Ser 25 Val G n Ser Lys Thr His Asp Lys IIe Leu Thr Val Cys G y Asn G y 40 Leu G y Thr Ser Leu Phe Leu Lys Asn Thr Leu G u G n Val Phe Asp 55 Thr Trp G y Trp G y Pro Tyr IVet Thr Val G u Al a Thr Asp Thr Ile 70 75 Ser Al a Lys G y Lys Al a Lys G u Al a Asp Leu Ile IVet Thr Ser G y 90 Su Ile Ala Arg Thr Leu y Asp Val y Ile Pro Val His Val lie 100 105 110 Asn Asp Phe Thr Ser Thr Asp G u Ile Asp Ala Ala Leu Arg G u Arg 115 120 125 Tyr Asp Ile 130 <210> 17 <211> 1827 <212> DNA <213> Cor ynebact eri um gl ut arri cum <220> <221> CDS <222> (101)..(1804) <223> RXN01244 <400> 17 gatatgtgtt tgtttgtcaa tatccaaatg tttgaatagt tgcacaactg ttggttttgt Page 18 seql i st cor r ect ed. t xt ggtgatcttg aggaaattaa ctcaatgatt gtgaggatgg gat Asp gt c Val caa Gn cgt Ar g t cc Ser ggc Gy aag Lys ttc Phe aca Thr gat Asp 150 gca Al a ttt Phe at c SIle ggc G y ct c Leu 230 gt c Val aat Asn t at Tyr ggc Gy gac Asp gct Al a gt c Val ggt Gy t cc Ser 120 ttg Leu gag Gu gac Asp gga G y gca Al a 200 aag Lys acc Thr gag G u gac Asp agc Ser gt c Val gct Al a gaa Gu gac Asp cct Pr o ttc Phe gac Asp ggt Gy t cc Ser 170 gt c Val cag G n at c SIle gac Asp ct c Leu 250 act Thr gcg Al a gt c Val gca Al a gga G y 75 cgt Ar g gcg Al a gaa G u at c SIle ct g Leu 155 cca Pr o act Thr ct c Leu aag Lys cgc Ar g 235 gag G u gt a Val gt g Val gcc Al a gcc Al a cca Pr o ggc Gy gaa G u gcc Al a cgc Ar g 140 cca Pr o gca Al a gag G u aac Asn t cc Ser 220 aac Asn cgc Ar g ct g Leu tgg Tr p gaa Gu 45 aca Thr gca Al a tgg Tr p t ac Tyr gca Al a 125 gac Asp gct Al a gac Asp ct g Leu gt g Val 205 ggc G y gcg Al a gct Al a aag Lys at t SIle gaa Gu gt c Val gct Al a cgt Ar g gcc Al a 110 ggc Gy cgc Ar g gtt Val acc Thr ggt G y 190 cct Pr o gaa G u gac Asp gct Al a ggc G y 15 acc Thr aac Asn tct Ser gag G u aag Lys 95 gt g Val ggc Gy gt c Val t cc Ser gcg Al a 175 ggc G y tgc Cys aag Lys gaa G u cgc Ar g 255 acc Thr cca Pr o cgt Ar g tct Ser gt g Val 80 gct Al a gtt Val ct g Leu at c SIle gga Gy 160 gca Al a cca Pr o at c SIle gt g Val gct Al a 240 at c SIle gtg gct act Val Al a Thr 1 ggc gtt gt c G y Val Val cgc ccc gaa Arg Pro G u gaa gca gag Su Al a u cgt ttg ctt Arg Leu Leu ctt aaa gct Leu Lys Al a gt c at c aag Val Ile Lys gca gca aca Al a Al a Thr 115 atc gcg gag Ile Ala G u 130 gca gaa ctt Al a G u Leu 145 cag gtc at t Sn Val lle ct a gac aca Leu Asp Thr acg agc cac Thr Ser Hi s 195 gt c gca t cc Val Al a Ser 210 ctt atc gac Leu Ile Asp 225 gaa gca acc G u Al a Thr gcc gag tgg Al a G u Tr p gt g Val ggt G y ct a Leu cag Gn gag G u act Thr ggt Gy 100 acc Thr cgc Ar g cgt Ar g ct c Leu gat Asp 180 acc Thr ggc G y ggc G y aag Lys aag Lys 260 gct Al a gga G y ccc Pr o gag Gu cgc Ar g gct Al a gtc Val aag Lys acc Thr ggc Gy ttt Phe 165 ct c Leu gcg Al a gcc Al a agc Ser ct c Leu 245 ggt Gy Page 19 seql i st cor r ect ed. t xt cct Pr o caa G n at c SIle cca Pr o 310 ttc Phe aag Lys ggt Gy cgc Ar g gac Asp 390 gaa G u ggc Gy at g let t ac Tyr gat Asp 470 gaa G u gca Al a ct g Leu caa G n ggc G y 280 ct g Leu gtt Val gag G u gtt Val cgt Ar g 360 ct c Leu gcc Al a aag Lys at g let cac Hi s 440 at g let tgg Tr p gct Al a cca Pr o gca Al a 520 aag gac ggc tac Lys Asp G y Tyr cgc gtt cag ctg ttg G n Leu Leu t ct Ser cgc Ar g gag G u aag Lys 330 ttc Phe ct g Leu gca Al a acc Thr ttt Phe 410 gaa G u gac Asp gcg Al a cca Pr o ttt Phe 490 ttg Leu t cc Ser gca Al a acc Thr cag G n 315 gt c Val gca Al a cgt Ar g at t l Ie tgg Tr p 395 gct Al a gtt Val ttt Phe gac Asp gca Al a 475 aac Asn gca Al a act Thr cag G n gaa G u 300 gct Al a gtt Val tcg Ser at c SIle gcg Al a 380 gtt Val gac Asp cca Pr o gtt Val cgc Ar g 460 gt c Val acc Thr act Thr gct Al a cag G n 285 ct g Leu gcg Al a gt c Val at g Met gca Al a 365 aag Lys at g Met at g Met gca Al a t cc Ser 445 at g Met ct g Leu ccg Pr o gt c Val ct c Leu 525 Ar g 270 gct Al a tgc Cys gt c Val cgc Ar g gct Al a 350 cgt Ar g gcc Al a gct Al a tgc Cys gca Al a 430 at c SIle tct Ser cgc Ar g gt c Val ct c Leu 510 gca Al a Val gca Al a ttc Phe t ac Tyr t cc Ser 335 gat Asp gga G y agc Ser cca Pr o cgt Ar g 415 t cc Ser ggt G y cct Pr o ct g Leu ggt G y 495 acc Thr gca Al a cag G n ctt Leu t ca Ser 320 ct c Leu gag G u cag G n gaa G u at g let 400 gag G u ct g Leu acc Thr gag G u at c SIle 480 gtt Val ggt G y gt c Val gaa G u 290 gcc Al a gt g Val gca Al a aac Asn gat Asp 370 ct c Leu gct Al a ggc G y gca Al a gac Asp 450 gcc Al a cac Hi s ggt G y ggc G y gca Al a 530 gcc Al a 275 gca Al a acc Thr ctt Leu ggt G y cca Pr o 355 ct g Leu ggc G y acc Thr ct a Leu gac Asp 435 ct g Leu t ac Tyr acc Thr gaa G u gt g Val 515 aag Lys aac Asn gaa G u gaa G u gaa G u tct Ser 340 gca Al a ct g Leu cgt Ar g gct Al a at c lie 420 aag Lys acc Thr ct g Leu tgt Cys gca Al a 500 aac Asn ct g Leu gt c Val ggc G y gag G u gca Al a 325 gac Asp ct g Leu act Thr ggc G y t at Tyr 405 gcc Al a at c SIe cag G n acc Thr gac Asp 485 gca Al a t cc Ser t ca Ser 931 979 1027 1075 1123 1171 1219 1267 1315 1363 1411 1459 1507 1555 1603 1651 1699 Page IO seql i st cor r ect ed. t xt gag gtc acc ctg gaa acc tgt aag aag gca gca gaa gca gca ctt gac 1747 SG u Val Thr Leu G u Thr Cys Lys Lys Ala Ala u Ala Ala Leu Asp 535 540 545 gct gaa ggt gca act gaa gca cgc gat gct gta cgc gca gtg atc gac 1795 L Ala u Gy Ala Thr u Ala Arg Asp Ala Val Arg Ala Val lie Asp 550 555 560 565 gca gca gtc taaaccactg ttgagctaaa aag 1827 Al a Al a Val 00 <210> 18 0 <211> 568 0 <212> PRT CK1 <213> Cor ynebact eri um gl ut arri cum 0 <400> 18 0 Val Ala Thr Val Ala Asp Val Asn G n Asp Thr Val Leu Lys y Thr C1 1 5 10 Sy Val Val Gy y Val Arg Tyr Ala Ser Ala Val Trp I e Thr Pro 25 Arg Pro u Leu Pro n Al a Gy Gu Val Val Ala G u u Asn Arg 40 G u Ala G u n G u Arg Phe Asp Ala Ala Ala Ala Thr Val Ser Ser 55 Arg Leu Leu G u Arg Ser G u Ala Ala G u y Pro Ala Ala G u Val 70 75 Leu Lys Ala Thr Ala G y Iet Val Asn Asp Arg Gy Trp Arg Lys Ala 90 Val lie Lys G y Val Lys G y G y His Pro Ala G u Tyr Ala Val Val 100 105 110 Al a Al a Thr Thr Lys Phe I e Ser IVet Phe G u Al a Al a G y y Leu 115 120 125 lie Ala u Arg Thr Thr Asp Leu Arg Asp lie Arg Asp Arg Val lie 130 135 140 Ala G u Leu Arg y Asp G u G u Pro y Leu Pro Ala Val Ser y 145 150 155 160 G n Val lie Leu Phe Ala Asp Asp Leu Ser Pro Ala Asp Thr Ala Ala 165 170 175 Leu Asp Thr Asp Leu Phe Val y Leu Val Thr G u Leu Gy y Pro 180 185 190 Thr Ser His Thr Ala lle Ile Ala Arg n Leu Asn Val Pro Cys Ile 195 200 205 Val Ala Ser y Ala y lie Lys Asp Ile Lys Ser y u Lys Val 210 215 220 Leu Ile Asp y Ser Leu y Thr Ile Asp Arg Asn Ala Asp u Ala 225 230 235 240 G u Ala Thr Lys Leu Val Ser u Ser Leu G u Arg Ala Ala Arg lie 245 250 255 Page 21 Ala O31u Tr p seqi i st car r ect ed. t xt Oy Pro Ala On Thr Lys Asp Oy Tyr Ar g Val O31n 270 Le u Thr Ser 305 Ly s As p vb t ValI O31u 385 ValI Ar g vb t As n Le u 465 Ly s Cys Le u O31y O31u 545 Ar g A a 275 A a Thr Le u O31y Pr o 355 Le u O31y Thr Le u As p 435 Le u Tyr Thr O31u ValI 515 Ly s A a ValI ValI O31y O31u A a 325 As p Le u Thr O31y Tyr 405 A a I Ie O31n Thr As p 485 A a Ser Ser As p As p 565 O31n I e Pr o 310 Ph e Ly s O31y Ar g As p 390 O31u O31y vb t Tyr As p 470 O31u A a Le u O31u A a 550 A a As p O31y 295 Ser Pr o Pr o ValI O31n 375 As p A a A a Pr a Thr 455 Pr a O31y As p Ser ValI 535 O31u A a O31y 280 Le u ValI O31u ValI Ar g 360 Le u A a Ly s vb t Hi s 440 vb t Tr p A a Pr a A a 520 Thr O31y ValI 265 As n Ph e As p Ser Pr a 345 O31y As p Pr a Tr p I Ie 425 Le u A a O31n Ar g Le u 505 A a Le u A a Ser Ar g O31u Ly s 330 Ph e Le u A a Thr Ph e 410 O31u As p A a Pr a Ph e 490 Le u Ser O31u Thr A a Thr O31n 315 ValI A a Ar g I Ie Tr p 395 A a ValI Ph e As p A a 475 As n A a Thr Thr O31u 555 O31n O31u 300 A a ValI Ser I Ie A a 380 ValI As p Pr a ValI Ar g 460 ValI Thr Thr A a Cys 540 A a O31n 285 Le u A a ValI vb t A a 365 Ly s vb t vb t A a Ser 445 vb t Le u Pr a ValI Le u 525 Ly s Ar g A a Cys ValI Ar g A a 350 Ar g A a A a Cys A a 430 li1e Ser Ar g ValI Le u 510 A a Ly s As p A a Ph e Tyr Ser 335 As p O31y Ser Pr a Ar g 415 Ser O31y Pr a Le u O31y 495 Thr A a A a A a <210O> 19 <21 1> 1629 <212> <213> <220> <221 Cor ynebact er i urn gI ut ani cumn ODS Page 22 seql i st cor r ect ed. t xt <222> (98)..(1606) <223> FRXA01244 <400> 19 agatgtcgat ttctcgagga agaagttaac gccgaagaaa accgtgaatc agagcaggag cgct t cgacg ccgctgcagc cacagtctct t ct t cgt ttg ctt gag cgc tcc gaa Leu Leu G u Arg Ser G u 1 gct Al a gt c Val ggt Gy t cc Ser ttg Leu gag G u gac Asp gga Gy gca Al a 135 aag Lys acc Thr gag G u caa G n ggc Gy 215 gaa G u gac Asp cct Pr o ttc Phe gac Asp ggt Gy t cc Ser 105 gt c Val cag G n at c SIle gac Asp ct c Leu 185 aag Lys tct Ser cca Pr o ggc Gy gaa G u gcc Al a cgc Ar g cca Pr o gca Al a gag G u aac Asn t cc Ser 155 aac Asn cgc Ar g ggc Gy cag G n gca Al a tgg Tr p tac Ty r gca Al a 60 gac Asp gct Al a gac Asp ct g Leu gt g Val 140 ggc Gy gcg Al a gct Al a tac Ty r cag G n 220 gct Al a cgt Ar g gcc Al a ggc Sy cgc Ar g gtt Val acc Thr ggt G y 125 cct Pr o gaa G u gac Asp gct Al a cgc Ar g 205 gct Al a gag G u aag Lys 30 gt g Val ggc Sy gt c Val t cc Ser gcg Al a 110 ggc G y tgc Cys aag Lys gaa G u cgc Ar g 190 gtt Val gca Al a gt g Val gct Al a gtt Val ct g Leu at c SIle gga G y gca Al a cca Pr o at c SIle gt g Val gct Al a 175 at c SIle cag G n cag G n ctt Leu gt c Val gca Al a at c SIle gca Al a 80 cag G n ct a Leu acg Thr gt c Val ctt Leu 160 gaa G u gcc Al a ct g Leu acc Thr aaa Lys at c SIle gca Al a gcg Al a gaa G u gt c Val gac Asp agc Ser gca Al a 145 at c SIle gca Al a gag G u ttg Leu gaa G u 225 gct Al a aag Lys aca Thr gag G u ctt Leu at t SIle aca Thr cac Hi s 130 t cc Ser gac Asp acc Thr tgg Tr p gcc Al a 210 gca Al a act Thr ggt Sy acc Thr cgc Ar g cgt Ar g ct c Leu gat Asp 115 acc Thr ggc G y ggc Sy aag Lys aag Lys 195 aac Asn gaa G u gct Al a gt c Val aag Lys acc Thr ggc G y ttt Phe 100 ct c Leu gcg Al a gcc Al a agc Ser ct c Leu 180 ggt Sy gt c Val ggc Sy ggc Sy aag Lys ttc Phe aca Thr gat Asp gca Al a ttt Phe at c SIe ggc G y ct c Leu 165 gt c Val cct Pr o caa G n at c SIe 115 163 211 259 307 355 403 451 499 547 595 643 691 739 787 ctg ttc cgc acc gaa ctg tgc ttc ctt tcc gcc Leu Phe Arg Thr G u Leu Cys Phe Leu Ser Ala acc gaa gag cca agc Thr G u G u Pro Ser Page 23 IO seql i st cor r ect ed. t xt o 235 240 245 gtt gat gag cag gct gcg gtc tac tca aag gtg ctt gaa gca ttc cca 883 Val Asp G u G n Ala Al a Val Tyr Ser Lys Val Leu G u Ala Phe Pro 250 255 260 d) [4 gag tcc aag gtc gtt gtc cgc tcc ctc gac gca ggt tct gac aag cca 931 Su Ser Lys Val Val Val Arg Ser Leu Asp Ala y Ser Asp Lys Pro 265 270 275 gtt cca ttc gca tcg atg gct gat gag atg aac cca gca ctg ggt gtt 979 Val Pro Phe Al a Ser IVet Al a Asp G u IVet Asn Pro Al a Leu G y Val 280 285 290 00 cgt ggc ctg cgt atc gca cgt gga cag gtt gat ctg ctg act cgc cag 1027 0 Arg y Leu Arg Ile Al a Arg Gy G n Val Asp Leu Leu Thr Arg G n S295 300 305 310 (N ctc gac gca att gcg aag gcc agc gaa gaa ctc ggc cgt ggc gac gac 1075 SLeu Asp Ala lle Ala Lys Ala Ser G u u Leu Gy Arg y Asp Asp 315 320 325 (N gcc cca acc tgg gtt atg gct cca atg gtg gct acc gct tat gaa gca 1123 Ala Pro Thr Trp Val bet Ala Pro bet Val Ala Thr Ala Tyr G u Ala 330 335 340 aag tgg ttt gct gac atg tgc cgt gag cgt ggc cta atc gcc ggc gcc 1171 Lys Trp Phe Ala Asp IVet Cys Arg Gu Arg y Leu lie Ala Gy Ala 345 350 355 atg atc gaa gtt cca gca gca tcc ctg atg gca gac aag atc atg cct 1219 IVet Ile G u Val Pro Ala Ala Ser Leu IVet Ala Asp Lys Ile IVet Pro 360 365 370 cac ctg gac ttt gtt tcc atc ggt acc aac gac ctg acc cag tac acc 1267 His Leu Asp Phe Val Ser IIe G y Thr Asn Asp Leu Thr G n Tyr Thr 375 380 385 390 atg gca gcg gac cgc atg tct cct gag ctt gcc tac ctg acc gat cct 1315 IVet Ala Ala Asp Arg IVet Ser Pro G u Leu Ala Tyr Leu Thr Asp Pro 395 400 405 tgg cag cca gca gtc ctg cgc ctg atc aag cac acc tgt gac gaa ggt 1363 Trp G n Pro Ala Val Leu Arg Leu IIe Lys His Thr Cys Asp G u G y 410 415 420 gct cgc ttt aac acc ccg gtc ggt gtt tgt ggt gaa gca gca gca gac 1411 Ala Arg Phe Asn Thr Pro Val y Val Cys Gy G u Ala Ala Ala Asp 425 430 435 cca ctg ttg gca act gtc ctc acc ggt ctt ggc gtg aac tcc ctg tcc 1459 Pro Leu Leu Ala Thr Val Leu Thr G y Leu G y Val Asn Ser Leu Ser 440 445 450 gca gca tcc act gct ctc gca gca gtc ggt gca aag ctg tca gag gtc 1507 Ala Ala Ser Thr Ala Leu Ala Ala Val Gy Ala Lys Leu Ser G u Val 455 460 465 470 acc ctg gaa acc tgt aag aag gca gca gaa gca gca ctt gac gct gaa 1555 Thr Leu G u Thr Cys Lys Lys Ala Ala G u Ala Ala Leu Asp Ala G u 475 480 485 ggt gca act gaa gca cgc gat gct gta cgc gca gtg atc gac gca gca 1603 Gy Ala Thr G u Ala Arg Asp Ala Val Arg Ala Val lie Asp Ala Ala 490 495 500 gtc taaaccactg ttgagctaaa aag 1629 Val Page 24 seql i st cor r ect ed. t xt <210> <211> 503 <212> PRT <213> Cor ynebact eri um gl ut am cum <400> Leu 1 Lys SIle Al a Al a G u Val Asp Ser Al a 145 I le Al a G u Leu G u 225 Al a Val Al a Asn Asp 305 Leu G u Arg Al a Thr Al a Lys G y Val Thr Thr Lys G u Arg Thr Leu Arg G y I e Leu Phe 100 Thr Asp Leu 115 Hi s Thr Al a 130 Ser G y Al a Asp G y Ser Thr Lys Leu 180 Trp Lys G y 195 Al a Asn Val 210 Al a G u G y Thr G u G u Leu G u Al a 260 G y Ser Asp 275 Pro Al a Leu 290 Ser 5 Sy Lys Phe Thr Asp Al a Phe l Ie Sy Leu 165 Val Pr o G n SIle Pr o 245 Phe Lys Sy G u Al a Al a u Val Sy Ser 55 Leu G u Asp Sy Al a 135 Lys Thr G u G n Sy 215 Leu Val G u Val Ar g 295 Asp Pr o Phe Asp Sy Ser 105 Val G n SIle Asp Leu 185 Lys Ser Ar g G u Lys 265 Phe Leu Sy 10 Ar g Al a Su I e Leu 90 Pr o Thr Leu Lys Ar g 170 G u Asp Al a Thr G n 250 Val Al a Ar g Pr o Sy G u Al a Ar g Pr o Al a G u Asn Ser 155 Asn Ar g Sy G n G u 235 Al a Val Ser SIle Al a Tr p Tyr Al a Asp Al a Asp Leu Val 140 Sy Al a Al a Tyr G n 220 Leu Al a Val lbt Al a 300 Al a Ar g Al a Sy Ar g Val Thr Sy 125 Pr o G u Asp Al a Ar g 205 Al a Cys Val Ar g Al a 285 Ar g Val Al a Val Leu SIe Sy Al a Pr o SIe Val Al a 175 l e G n G n Leu Ser 255 Leu G u G n Leu Leu Thr Arg n Leu Asp Ala lie Ala Lys Ala Ser u u 310 315 320 Page seql i st cor r ect ed. t xt Leu G y Arg G y Asp Asp 325 Al a Pro Thr Al a Gy Al a Asp 385 Al a Hi s Gy Gy Al a 465 Al a Al a Al a SIle 355 Lys Thr Leu Cys Al a 435 Asn Leu Leu l Ie G u Gy bet Tyr Asp 405 G u Al a Leu G u Al a 485 Al a Al a Al a Pr o Thr 390 Pr o Gy Asp Ser Val 470 G u Al a Lys lbet Hi s 375 lbt Tr p Al a Pr o Al a 455 Thr Gy Val Tr p SIle 360 Leu Al a G n Ar g Leu 440 Al a Leu Al a Phe 345 G u Asp Al a Pr o Phe 425 Leu Ser G u Thr Tr p 330 Al a Val Phe Asp Al a 410 Asn Al a Thr Thr G u 490 Asp Pr o Val Ar g 395 Val Thr Thr Al a Cys 475 Al a Val et Ala Pro let Val Cys Al a 365 I le Ser Ar g Val Leu 445 Al a Lys Asp Ar g 350 Ser Gy Pr o Leu Gy 430 Thr Al a Al a Al a 335 G u Leu Thr G u SIe 415 Val Gy Val Al a Val 495 <210> 21 <211> 390 <212> DNA <213> Corynebacter i um gl ut ari cum <220> <221> CDS <222> (101)..(367) <223> RXA01300 <400> 21 gatcgacatt aaatcccctc ccttgggggg tttaactaac gttcggatta acggcgtagc aacacgaaag gacactttcc gt a Val at c SIle gtt Val at c gt c Val gaa Gu t cc Ser gcg t cc Ser gct Al a gat Asp ggc t cc Ser gct Al a gac Asp gca ct g Leu gac Asp acc Thr ggc cac gca Hi s Al a 15 gac gaa Asp u gac gcg Asp Al a aac gaa Page 26 aaatcgctgc gccctaatcc atg gct tcc aag act IVet Ala Ser Lys Thr 1 cgt cca gca tcc atc Arg Pro Ala Ser lie atc ttg ctg acc ctg I e Leu Leu Thr Leu tcc tct tcc ctc atg Ser Ser Ser Leu IVet gtt acc gtc acc tcc I I e I'b t Al a gac aac gct Asp Asn Al a ct t gac gct Leu Asp Al a seqI i st car r ect ed. t xt Leu(1y Al a 1u Hi s 1y Asn (1u Val ThrVal ThrSer 60 gaa gct gt t gag aag at c gct gcg ct t at c gca cag gac 31u Al aVa 1G31u Lys lleAlaAla Leu lleAla(3n Asp 75 80 gag taaacaacgc t ct gct tgt t aaa (31u <210O> 22 <21 1> 89 <21 2> PRT <21 3> Cor ynebact er i urn gi ut anri curn <400> 22 I'b t Al a Ser Lys Thr Val Thr Val G31y Ser Ser Val G31y Leu Hi s Al a 1 5 10 Ar g Pro Al a Se r I Ie Ile Ala Ou AlaAla Al a (1u Ty r As p As p G31u 25 li1e Leu Leu Thr Leu Val G31ySe r As pAs pAs p (1u 1u Thr As pAl a 40 Ser Ser Ser Leu 'b t I Ie I'b t Al a Leu G31y Al a G31u Hi s G31y As n G31u 55 Val Thr Val Thr Ser As p As n Al a G31u Al a Val G31u Lys I Ie Ala Ala 70 75 Leu I I e Al a G3 n As p Leu As p Al a G3 u <210O> 23 <21 1> 508 <212> DNA <213> Cor ynebact er i urn gi ut ani cumn <220> <221 CIDS <222> (508) <223> RXN03002 <400> 23 ggaact t cga ggt gt ct t cg t ggggcgt ac ggagat ct ag accct at ccg aat caacat g cagt gaat ta acat ct act t g at As p t gg Tr p ag c Ser aaa ct a Le u g aa G31u g at As p ccc Pr o gca Al a at c I Ie gcc Al a at t I Ie gaa G31u cgc Ar g t ac Tyr gt g ValI cgc Ar g gcc Al a ac c Th r gt c ValI at a I Ie gca Al a g at As p 45 gct Al a g aa G31u ggt G31y gcc Al a cca Pr o ct C gac Leu Asp 15 gt a ct c Val Leu at g at c I'b t I Ie ggt tt c G3 y Phe Page 27 caagt gt ggc t t tatgt t tg at g t tt g ta ct c aaa 115 I'b t Phe Val Leu Lys 1 cgc acg gtc acc gat 163 Ar g Thr Val Thr Asp cta gaa aag aca aac 211 Leu G3 u Lys Thr Asn gcc agc gtg gaa gaa 259 Al a Ser Val a31u a31u gct ttc gcg cac gcc 307 Al a Phe Al a Hi s Al a seql i 60 cgc ccc agc aga gca gtc cgc gag acc Arg Pro Ser Arg Ala Val Arg G u Thr 75 gcc tcc cct gtt tcc ttc ggt cac agt Ala Ser Pro Val Ser Phe G y His Ser atc gtt gct ctc gct gcc aaa gat gcc Ile Val Ala Leu Ala Ala Lys Asp Ala 105 110 gcg gca ttg gct aaa gct tta gga aaa Al a Al a Leu Al a Lys Al a Leu G y Lys 120 125 gca caa agt Al a G n Ser 135 <210> 24 <211> 136 <212> PRT <213> Cor ynebact eri um gl ut ari cum <400> 24 IVet Phe Val Leu Lys Asp Leu Leu Lys 1 5 Arg Thr Val Thr Asp Trp Arg G u y 25 Leu G u Lys Thr Asn Ser Ile Asp Ser 40 Ala Ser Val G u G u Lys y Pro Tyr 55 Ala Phe Ala His Ala Arg Pro Ser Arg 70 Ser Trp Val Arg Leu Ala Ser Pro Val Asp Pro Leu Asn Leu Ile Val Ala Leu 100 105 Hi s Thr G n Al a Met Al a Al a Leu Al a 115 120 Lys Asp Leu Asp G u Al a G n Ser 130 135 <210> <211> 789 <212> DNA <213> Cor ynebact eri um gl ut ai cum <220> <221> CDS <222> (14)..(766) <223> RXC00953 <400> st cor r ect ed. t xt gct atg tcg tgg Ala Vet Ser Tr p 80 aag aat gat ccc Lys Asn Asp Pro 95 acc gca cat acc Thr Ala His Thr t ac cga aag gat Tyr Arg Lys Asp 130 gtg cgc ctg Val Arg Leu ctc aat ct c Leu Asn Leu 100 caa gcg atg G n Ala vet 115 ctc gac gag Leu Asp G u Al a 10 SIle Al a SIle Al a Ser 90 Al a Lys Ar g Al a Thr Val Ar g Sy Lys Leu SIle Al a Asp Al a G u Hi s Asp Sy 125 G u Sy Al a Pr o Thr Ser Al a 110 Lys Leu Val Met Sy Al a Lys Thr Tyr Asp Leu SIe Phe bet Asn Al a Ar g Page 28 seqli cttgcattcc cca atg gcg cca cca acg l'bt Ala Pro Pro Thr st car r ect ed. t xt gt a ggc aac t ac at c at g cag t cc 52 Val G31y Asn Tyr I Ie l'bt O31n Ser tt c Ph e ggt G31y gct Al a gt g ValI tt c Ph e cca Pr o tt c Ph e 110 cga Ar g ct c Le u ac c Thr t ct Ser at c I Ie 1 90 gt g ValI gcg Al a at t I Ie caa ggt (31n (1y cgc acc Ar g Thr aag gtt Lys Val ccc t ac Pr o Ty r ggt ggc G31YG(1Y t tt ggt Phe G3 y ggt ggc G31YG(1Y gca gt a Al a Val gct tt c Al a Phe 145 t tc ggt Phe 03 y 1 60 gcc aag Al a Lys gcg gtt Al a Val ggg cac 03 y Hi s gct gat Al a As p 225 cct ccg Pr o Pr o ct g Le u at t I Ie gt t ValI gcg Al a tt g Le u gt c ValI gcg Al a tt t Ph e 130 ct g Le u g at As p gt g ValI ct t Le u t gg Tr p 210 gcc Al a gcg Al a cag (31n ct t Le u 35 ccc Pr o cag O31n gt t ValI gcg Al a gcg Al a 115 ggc G31y ct t Le u gcg Al a gaa O31u ct g Le u 195 g at As p act Thr ggc G31y tt c Ph e ggt G31y gga G31y aac As n ggc G31y tt g Le u 1 00 ggc G31y gcc Al a ggt G31y g ac As p ggt G31y 1 80 ggt G31y cca Pr o cca Pr a gct Al a ggc (31y g aa O31u gct Al a gcc Al a ct g Le u 85 at t I Ie gt t ValI tt t Ph e gt g ValI tt t Ph e 1 65 gct Al a ggc G31y gct Al a ac g Thr gt t ValI ct g Le u at c I Ie gt t ValI 70 act Thr ct g Le u t ac Tyr gcc Al a ct t Le u 1 50 ggt G31y ggc G31y gcg Al a ccc Pr a gct Al a 230 gca Al a gt c ValI ccc Pr a 55 ct c Le u gt t ValI cct Pr a ggt G31y aac As n 135 ggt G31y t gg Tr p ggg G31y at g vb t aac As n 215 ggg G31y gt t ValI ccc Pr a gca Al a at t I Ie ct t Le u ggt G y aat As n 120 ggt G3y t cc Ser tt c Ph e ct c Le u gt c ValI 200 cgt Ar g gct Al a gcc Al a gca Al a tt g Le u ggt G31y gca Al a tt g Le u 1 gcc Al a ct t Le u tt c Ph e gga G31y at c I Ie 1 tt c Ph e gag O31u Ar g gt g ValI tt c Ph e g at As p tt c Ph e t cg Ser gt c ValI ac g Thr ct g Le u ggg G31y at c I Ie 1 tt g Le u c ag O31n cgc Ar g ac c Thr at t I Ie c aa O31n gca Al a tt g Le u t gg Tr p ccc Pr a ggt O31y at t I Ie t ca Ser 1 gt t ValI tt g Le u aag Ly s gt g ValI t ac Tyr 235 ct c Le u ggt O31y ccg Pr a t ct Ser ct g Le u c ac Hi s ggt O31y ac c Thr 140 gag O31u gt t ValI ct c Le u cgc Ar g gag O31u 220 cct Pr a cct acc cca Pro Thr Pro 245 ccg gct cga agc Pro Ala Arg Ser 250 240 t aagat ct cc aaaaccct ga gat <210O> 26 Page 29 seqi i st car r ect ed. t xt <21 1> 251 <21 2> PRT <21 3> Cor ynebact er i urn gi ut ani cumn <400> 26 [vb t A a Pro Pro Thr Val Gy Asn Tyr G31y Thr ValI Tyr G31y G31y G31y ValI Ph e 145 G31y Ly s ValI Hi s As p 225 Pr o O31n Le u Pr a O31n ValI A a A a 115 G31y Le u A a O31u Le u 1 95 As p Thr G31y G31y O31u A a A a Le u li1e ValI Ph e ValI Ph e 1 65 A a G31y A a Th r Pr a 245 ValI Le u I Ie ValI 70 Thr Le u Tyr A a Le u 150 G31y G31y A a Pr a A a 230 Thr A a ValI Pr a Le u ValI Pr a G31y As n 135 G31y Tr p G31y vb t As n 215 G31y Pr a ValI Pr a 40 A a I Ie Le u G31y As n 120 G31y Ser Ph e Le u ValI 200 Ar g A a Pr a A a A a Le u G31y A a Le u 1 05 A a Le u Ph e Gy le 1 85 Ph e O31u Ar g A a O31n A a Le u 75 Tr p Pr a G31y I Ie Ser 1 55 ValI Le u Ly s ValI Tyr 235 Ser Ser Ph e I Ie li1e Ser As n Ph e Ar g 125 Ph e As n Gy le ValI 205 Ly s Ly s Ph e G31y A a ValI Ph e Pr a Ph e 110 Ar g Le u Thr Ser I Ie 1 ValI A a I Ie Thr ValI A a Ph e ValI A a Thr G31y Pr a Thr A a 1 A a As n (31u A a (31n Ar g Ly s Pr a G31y Ph e G31y A a A a Ph e 1 A a A a G31y A a Pr a 240 <210O> 27 <21 1> 553 <212> DNA <213> Cor ynebact er i urn gi ut ani cumn <220> <221 ODS <222> (553) <223> RXC03001 <400> 27 cccggt t cac gt gat caat g act t cacgag caccgat gaa at cgat gct g cgct t cgt ga acgct acgac at ct aact ac t t taaaagga cgaaaat at t at g gac t gg t ta acc 115 Page seqI i st car r ect ed. t xt I'b t As p Tr p Leu Thr at t cct I Ie Pr o at c ggt I Ie G31y ggt cag (31y (1n at t ggt I Ie G31y gcg at g A a [Vb t gaa gcc (3 u A a t gg ct g Trp Leu acc aac Thr As n t gc acc Cys Thr 135 ct t Le u at c I Ie gt t ValI gcg A a at c I Ie at c I Ie at g vb t ct g Le u 120 ct c Le u ac c Thr ggt G31y gcc A a ggt G31y gga G31y ct g Le u t at Tyr gt t ValI gcc A a gga G31y ac g Thr gcc A a 75 at c G31y gt c ValI aat As n gt g ValI gca A a tt g Le u ac a Thr gca A a tt c Ph e tt g Le u gt c ValI 140 g aa O31u gga G31y at c lIe 45 gt c ValI ggc G31y c ag O31n gcc A a ct c Le u 125 at c I Ie tt g Le u aaa Ly s act Thr at g vb t gct A a at c I Ie 110 aac As n ct t Le u 15 ggt G31y gca A a gcc A a cgt Ar g g aa O31u 95 t ct Ser gga G31y gcg A a gcc A a ac g Thr t cc Ser ggt G31y 80 t ac Tyr tt g Le u c ac Hi s ccg Pr o ggg G31y G31y gag O31u gt c ValI gcg A a tt g Le u gt g ValI 130 gct A a cgt Ar g tt t Ph e cca Pr o cca Pr o c ag O31n gct A a 115 ct g Le u tt c Ph e t cc Ser tt g Le u ct g Le u ac g Thr gt g ValI 1 00 cgt Ar g tt g Le u ct c Le u gt c ValI ct c Le u ggt G31y aat As n gcg A a tt c Ph e at g vb t at g ct c acc at g I'bt Leu Thr I'bt ttg gcc acc gga Leu A a Thr 03 y at c tt c I I e Phe 1 <210O> 28 <211> 151 <21 2> PRT <21 3> Cor ynebact er i urn gi ut ani cumn <400> 28 I'bt Asp Tr p Leu Thr I Ie Pro Leu Phe 1 5 Val Pro Ala Phe Leu lieGy Ilie Ilie I'b t G31y Ar g Se r Val Gy On Val I Ie 40 Leu 03 y Phe Leu Leu I I e 03 y A a 03 y 55 Leu O31u Pr o Leu G31y Ala 'b t I Ie I'b t 70 Val Val Pr o Thr Asn Ou Ala Ilie Ala Leu Val 10 Thr A a G31y (1y A a Thr O3 y A a G31y I Ie 90 Page 31 ag a Ar g 145 As n ValI A a Le u Thr A a gt t gat gcg t gg Val Asp Ala Trp (31u Gy le ValI G31y O31n Le u O31y A a A a Ar g O31u seqI i st car r ect ed. t xt Gy Ala On Val Ala Trp Leu I'bt Ilie Leu Gy Phe Ala lieSer Leu 100 105 110 Val Leu Al a Ar g Phe Thr As n Leu Ar g Tyr Val Leu Leu As n G31y Hi s 115 120 125 Hi s Val Leu Leu I'b t Cy s Thr I'b t Leu Thr I'bt Val Leu Al a Th r 03 y 130 135 140 Ar g Val As p Al a Tr p I Ie Phe 145 150 <210O> 29 <21 1> 2172 <21 2> DNA <21 3> Cor ynebact er i urn gi ut anri cumn <220> <221 OIDS <222> (2149) <223> RXN01943 <400> 29 ccgatt ct tt t tcggcccaa t tcgt aacgg cgat cct ct t aagt ggacaa gaaagt ct ct t gcccgcggg agacagaccc t acgt t taga aaggt t tgac at g gcg t cc I'bt Al a Ser ac g Thr at t I Ie g at As p ct t Le u gga G31y aag Ly s t ac Tyr tt c Ph e t ca Ser ac a Thr t cg Ser t cc Ser gt a ValI gt t ValI tt c Ph e gga G31y t ct Ser 120 at t I Ie c aa O31n act Thr gt t ValI ccc Pr o aac As n g ac As p cgt Ar g act Thr ac c Thr at t I Ie t gt Cys c aa O31n gga G31y t ac Tyr g aa (31u aag Ly s cga Ar g tt g Le u 140 ct g Le u gcg Al a c aa O31n 45 t cc Ser c aa O31n gct Al a t ac Tyr cca Pr o 125 gt t ValI g aa O31u act Thr g aa O31u ac c Thr g aa O31u ac a Thr t cg Ser 110 at c I Ie ct t Le u aac As n 15 cgc Ar g at t I Ie ggt O31y at c I Ie gag O31u 95 t gg Tr p ct g Le u gcg Al a ct t Le u ct t Le u g ac As p at g vb t ct c Le u 80 agt Ser at t I Ie t gg Tr p g at As p g at As p 1 ggt O31y cgc Ar g t cc Ser c ag O31n aaa Ly s t ca Ser g ac As p gcc Al a act Thr 145 gga O31y tt c Ph e g ac As p gt g ValI ct t Le u t cc Ser t ac Tyr ct g Le u 130 tt c Ph e cca Pr o c aa O31n cca Pr o gt g ValI g at As p aag Ly s gcc Al a 115 ct t Le u ggt O31y aaa ct g Lys Leu gac aat As p As n gt g aag Val Lys t ca gt t Ser Val at g ggt [Vb t (1y gga at g 31y Ib t aag gaa Lys 03 u 1 00 ttc gag Phe 03 u ggt gcc O3 y Al a ttg caa Leu 03 n gac t t c cgc gct cca at g gat gag cag cct Asp Phe Arg Ala Pro I'bt Asp Ou On Pro act tat gta ttc ctg Thr Tyr Val Phe Leu Page 32 seql i st cor r ect ed. t xt 150 cac Hi s gcc Al a at t SIle ggc Gy t cc Ser 230 gt g Val ttc Phe ct g Leu ctt Leu cca Pr o 310 aac Asn cag G n ggc Gy gtt Val cct Pr o 390 ct c Leu at g Met gca Al a gcc Al a 200 acc Thr cag G n aag Lys cca Pr o gga Gy 280 gcg Al a ct c Leu at c SIle cca Pr o ttc Phe 360 ct g Leu ct c Leu ccg Pr o cgc Ar g 170 cga Ar g ctt Leu aca Thr ttc Phe ct g Leu 250 ttc Phe ttc Phe aac Asn cca Pr o at c SIle 330 ggt Gy ct c Leu ggc Gy ggt Gy tgt Cys 410 tcg Ser aag Lys ctt Leu gt c Val cca Pr o 235 aag Lys t cc Ser ggc Gy aac Asn tt c Phe 315 cag G n gcc Al a t cc Ser at g bet gtt Val 395 tt g Leu gt c Val ct c Leu act Thr ttt Phe 220 ccg Pr o aag Lys ct g Leu at c SIle ttc Phe 300 ttg Leu aac Asn tgg Tr p at t SIle ttg Leu 380 ct g Leu gca Al a t ac Tyr gca Al a 190 gaa G u ct g Leu at t SIle at c SIle at t SIle 270 gtt Val cca Pr o cca Pr o aac Asn ttc Phe 350 gaa G u ggt Gy cga Ar g ggt Gy ttc Phe 175 aac Asn ttc Phe cca Pr o gca Al a cct Pr o 255 at g Met ggt Gy ttt Phe ctt Leu acc Thr 335 gcc Al a cga Ar g ttg Leu ttc Phe at c SIle 415 160 ct g Leu gag G u ttg Leu at g Met gca Al a 240 gaa G u at c SI e aac Asn at t SIle gga Gy 320 ct g Leu tgc Cys aac Asn ct c Leu aag Lys 400 gt g Val cca Pr o tgg Tr p gca Al a gtt Val 225 at t l Ie gca Al a cca Pr o gga Gy ctt Leu 305 ttg Leu ggt Gy ttc Phe aag Lys ggc Gy 385 aag Lys at g Met at t SIle at t SIle ct g Leu 210 ct g Leu ggt Gy gt c Val gcg Al a at t SIle 290 t cc Ser cac Hi s t ac Tyr ggc Gy gcc Al a 370 ggc Gy acc Thr ggc Gy gtt Val 180 gca Al a tct Ser gac Asp t ac Tyr at g Met 260 gca Al a aac Asn gtt Val cca Pr o ttc Phe 340 gt c Val cgt Ar g t cc Ser ttc Phe ttc Phe 420 ggt Gy gct Al a gcc Al a t ac Tyr tgg Tr p 245 gt g Val ttc Phe ct g Leu at c SIe ct a Leu 325 at t SIe acc Thr cag G n gag G u cgc Ar g 405 gac Asp gca Al a 643 691 739 787 835 883 931 979 1027 1075 1123 1171 1219 1267 1315 1363 1411 atc aag gcg tac gct ttc gtg ttc acc tcc Ile Lys Ala Tyr Ala Phe Val Phe Thr Ser ttg ctt acc atc cca Leu Leu Thr lie Pro Page 33 seql i st cor r ect ed. t xt at g let gtt Val cgc Ar g 470 gat Asp gca Al a acc Thr at t SIle gac Asp 550 cca Pr o gt c Val gaa G u ggc Gy cca Pr o 630 ttg Leu gaa G u gt c Val cca Pr o 440 at g let gag G u aag Lys gcg Al a gt g Val 520 t cc Ser at c SIle gga Gy aaa Lys ctt Leu 600 acc Thr at c SIle acc Thr at t SIle ggc Gy 680 ttg Leu ctt Leu cgt Ar g gaa G u 490 gcc Al a gct Al a ct c Leu gca Al a acc Thr 570 gga Gy cac Hi s cac Hi s ttt Phe gtt Val 650 gca Al a ggc Gy gtt Val gca Al a 475 gct Al a ggt Gy aag Lys gaa G u gca Al a 555 gtt Val cac Hi s gtt Val gtt Val gac Asp 635 gt g Val gat Asp t ac Tyr ct c Leu 460 aag Lys aat Asn gca Al a ccg Pr o ggc Gy 540 ggc Gy gtt Val gca Al a gga Gy gag G u 620 gct Al a gt g Val cag G n acc Thr 445 gca Al a gtt Val gca Al a ggt Gy aag Lys 525 aag Lys aag Lys gct Al a gt g Val ttg Leu 605 cgc Ar g gac Asp tct Ser gca Al a 430 at t SIle ct g Leu gct Al a act Thr gca Al a 510 ct g Leu gca Al a ctt Leu cca Pr o gca Al a 590 gac Asp agg Ar g ttc Phe aac Asn aat Asn 670 ggt Gy gac Asp gct Al a cct Pr o 495 gga Gy gcc Al a at t l Ie gga Gy gca Al a 575 ttg Leu acc Thr cag G n at t SIle gcc Al a 655 tct Ser at c SIle t ac Tyr gac Asp 480 gca Al a gcc Al a gct Al a cca Pr o cca Pr o 560 gac Asp cgc Ar g gt g Val caa G n cga Ar g 640 gcg Al a t cc Ser gca Al a cgt Ar g 465 aag Lys gct Al a gct Al a ggg Gy ctt Leu 545 ggc Gy gct Al a tta Leu caa G n gt c Val 625 t cc Ser aaa Lys acg Thr gtt Val 450 t cc Ser cag G n cca Pr o gct Al a gaa G u 530 tct Ser at t SIle act Thr gat Asp ttg Leu 610 aag Lys aag Lys ttc Phe act Thr gca Al a aac Asn gca Al a gt a Val ggc Sy 515 gt a Val gaa G u gca Al a gt c Val agc Ser 595 ggc G y gcg Al a gat Asp ggt Sy gt g Val 675 ttc Phe gaa G u gaa G u gct Al a 500 gct Al a gt g Val gt a Val at c l Ie at c SIle 580 gga G y ggc G y ggg Sy ct a Leu gaa G u 660 at c SIle ttc Phe gag G u gaa G u 485 gct Al a gca Al a gac Asp cct Pr o caa G n 565 ctt Leu gtt Val gaa G u gat Asp cct Pr o 645 at t SIe aag Lys 1459 1507 1555 1603 1651 1699 1747 1795 1843 1891 1939 1987 2035 2083 2131 2172 aag aac gag Lys Asn G u t aacct ggga t ccat gt t gc gca <210> Page 34 seqI i st car r ect ed. t xt <21 1> 683 <21 2> PRT <21 3> Cor ynebact er i urn gi ut anm cumn <400> N/t A a Ser Lys Leu Thr Thr Thr Ser Ar g Ser O31n Ly s Ser As p A a Thr 145 Thr Pr a Tr p A a ValI 225 li1e A a Pr a O31y Le u 305 Pr a O31n Pr a ValI As p Ly s A a 115 Le u O31y ValI vb t O31y 1 95 O31y As n Le u O31n Thr 275 Ser I Ie As n Ly s ValI G31y vb t (31u (31u A a O31n Le u 1 65 O31y A a A a Tyr Tr p 245 ValI Ph e Le u I Ie Thr O31n O31y Ser Hi s O31y Le u Le u 135 Ph e Ser Thr Pr a As p 215 O31y O31u ValI Le u O31u 295 Le u Ser Ser 40 ValI ValI Ph e O31y Ser 120 li1e Ar g vb t A a A a 200 Thr O31n Ly s Pr a O31y 280 A a Le u vb t li1e ValI A a A a ValI 1 As p I Ie A a Tr p A a 1 A a ValI ValI O31y Ph e 265 Pr a I Ie Tyr Hi s Hi s As p O31n Tyr 75 O31y O31y Ph e Le u vb t 1 55 Ser Ly s Le u ValI Pr a 235 Ly s Ser O31y As n Ph e 315 Le u A a O31n Ser O31n A a Tyr Pr a 125 ValI O31u Ph e O31y Pr a 205 O31y Le u I Ie Le u O31y 285 Ser ValI As n Ar g I Ie Oy le Ou Tr p Le u A a Pr a Ph e 1 As n Ph e Pr a A a Pr a 255 vb t O31y Ph e Le u Le u Le u As p vb t Le u Ser I Ie Tr p As p As p 1 Le u O31u Le u vb t A a 240 Ou le As n I Ie O31y 320 Leu His Trp Pro LeuAsnAia Ile I'bt Iie(3nAsn IieAsnThr Leu 325 330 335 Page seqI i st car r ect ed. t xt Gy Tyr Asp Phe Ilie On Gy Pro I'bt Gy Ala Trp Asn Phe A a Cys 350 340 Ph e Ly s G31y 385 Ly s vb t Le u A a Ar g 465 Ly s A a A a G31y Le u 545 O31y A a Le u O31n ValI 625 Ser Ly s Thr Le u 355 vb t I Ie Tyr I Ie I Ie 435 A a As n A a ValI O31y 515 ValI O31u A a ValI Ser 595 O31y A a As p G31y ValI 675 Thr O31n (31u Ar g 405 As p A a Ph e (31u (31u 485 A a A a As p Pr o O31n 565 Le u ValI O31u As p Pr o 645 li1e Ly s O31y ValI Pr o 390 Le u I Ie vb t ValI Ar g 470 As p A a Thr I Ie As p 550 Pr o ValI O31u O31y Pr a 630 Le u O31u ValI ValI Ser 375 Ser Le u Ly s As p Ser 455 As p Le u O31y A a ValI 535 Pr a Thr O31n I Ie Ph e 615 Le u I Ie O31y As n Ph e 360 Le u Le u Pr a A a Pr a 440 vb t O31u Ly s A a ValI 520 Ser I Ie O31y Ly s Le u 600 Thr I Ie Thr I Ie O31y 680 345 Le u O31y Tyr O31y Tyr 425 Tr p Ph e A a A a O31y 505 A a Pr a Ph e As n Ser 585 ValI ValI Thr Pr a Pr a 665 Ly s Le u O31y O31y Cys 410 A a Le u Le u Ar g O31u 490 A a A a Le u A a Thr 570 O31y Hi s Hi s Ph e ValI 650 A a As n Ser vb t ValI 395 Le u Ph e O31y ValI A a 475 A a O31y Ly s O31u A a 555 ValI Hi s ValI ValI As p 635 ValI As p O31u I Ie Le u 380 Le u A a ValI Tyr Le u 460 Ly s As n A a Pr a O31y 540 O31y ValI A a O31y O31u 620 A a ValI O31n Ly s 365 A a Le u O31y Ph e Thr 445 A a ValI A a O31y Ly s 525 Ly s Ly s A a ValI Le u 605 Ar g As p Ser A a O31u O31y Ar g O31y Thr 430 li1e Le u A a Thr A a 510 Le u A a Le u Pr a A a 590 As p Ar g Ph e As n As n 670 Ar g Le u Ph e I Ie 415 Ser O31y As p A a Pr a 495 O31y A a I Ie O31y A a 575 Le u Thr O31n I Ie A a 655 Ser As n Le u Ly s 400 ValI Le u I Ie Tyr As p 480 A a A a A a Pr a Pr a 560 As p Ar g ValI O31n Ar g 640 A a Se r <210O> 31 Page 36 seql i st cor r ect ed. t xt <211> 1339 <212> DNA <213> Corynebacter i um gl ut ai cum <220> <221> CDS <222> (101)..(1339) <223> FRXA02191 <400> 31 ccgattcttt ttcggcccaa ttcgtaacgg cgatcctctt tgcccgcggg agacagaccc tacgtttaga aaggtttgac acg Thr at t SIle gat Asp ctt Leu gga Gy aag Lys t ac Tyr ttc Phe t ca Ser gac Asp 150 cac Hi s gcc Al a at t SIle aca Thr tcg Ser t cc Ser gt a Val gtt Val ttc Phe gga Gy tct Ser 120 at t SIle cgc Ar g at g Met gca Al a gcc Al a 200 caa G n act Thr gtt Val ccc Pr o aac Asn gac Asp cgt Ar g act Thr acc Thr cca Pr o cgc Ar g 170 cga Ar g ctt Leu at t SIle tgt Cys caa G n gga Gy t ac Tyr gaa G u aag Lys cga Ar g ttg Leu 140 gat Asp gt c Val ct c Leu act Thr ct g Leu gcg Al a caa G n 45 t cc Ser caa G n gct Al a t ac Tyr cca Pr o 125 gtt Val gag G u ttc Phe ggc Gy cca Pr o 205 gaa G u act Thr gaa Gu acc Thr gaa G u aca Thr tcg Ser 110 at c SIle ctt Leu cag G n t ac Tyr gca Al a 190 gaa G u ctt Leu ctt Leu gac Asp at g Met ct c Leu 80 agt Ser at t SIle tgg Tr p gat Asp gat Asp 160 ct g Leu gag G u ttg Leu aagt ggacaa gaaagt ct ct atg gcg tcc aaa ctg 11! IVet Ala Ser Lys Leu 1 ggt gga cca gac aat 16 G y G y Pro Asp Asn cgc ttc caa gtg aag 21 Arg Phe G n Val Lys tcc gac cca tca gtt Ser Asp Pro Ser Val cag gtg gtg atg ggt G n Val Val Met y aaa ctt gat gga atg Lys Leu Asp G y IVet tca tcc aag aag gaa Ser Ser Lys Lys G u 100 gac tac gcc ttc gag Asp Tyr Ala Phe G u 115 gcc ctg ctt ggt gcc 49! Al a Leu Leu G y Al a 130 act ttc ggt ttg caa 54 Thr Phe y Leu G n 145 act tat gta ttc ctg 59! Thr Tyr Val Phe Leu 165 cca att atg gtt ggt 64; Pro Ile IVet Val y 180 tgg att ggt gca gct 69 Trp lie y Ala Ala 195 gca ctg ggt tct gcc 73! Al a Leu G y Ser Ala 210 Page 37 ggc gat Sy Asp 215 tcc gga Ser G y 230 gtg gaa Val G u ttc gtc Phe Val ctg ctt Leu Leu ctt gaa Leu G u 295 cca ttg Pro Leu 310 aac gcc Asn Ala cag gga G n y ggc gtg G y Val gtt tcc Val Ser 375 cct t cc Pro Ser 390 ct c ct g Leu Leu acc gtc aca gtc ttt gg Thr Val Thr Val Phe G 220 seql i st cor r ect ed. t c ctg cca atg gtt v Leu Pro IVet Val ctg aat gac tac Leu Asn Asp Tyr cag G n aag Lys cca Pr o gga Gy 280 gcg Al a ct c Leu at c l Ie cca Pr o ttc Phe 360 ct g Leu ct c Leu ccg Pr o ttc Phe ct g Leu 250 ttc Phe ttc Phe aac Asn cca Pr o at c SIle 330 ggt Gy ct c Leu ggc Gy ggt Gy tgt Cys 410 cca Pr o 235 aag Lys t cc Ser ggc Gy aac Asn tt c Phe 315 cag G n gcc Al a t cc Ser at g bet gtt Val 395 tt g Leu ccg Pr o aag Lys ct g Leu at c SIle ttc Phe 300 ttg Leu aac Asn tgg Tr p at t SIle ttg Leu 380 ct g Leu gca Al a ct g Leu at c SIle ct g Leu ggt Gy 285 agc Ser gtt Val at c SIle aac Asn aag Lys 365 gct Al a ct c Leu gca Al a at t SIle at c SIle at t SIle 270 gtt Val cca Pr o cca Pr o aac Asn ttc Phe 350 gaa G u ggt Gy cga Ar g gca Al a cct Pr o 255 at g Met ggt Gy ttt Phe ctt Leu acc Thr 335 gcc Al a cga Ar g ttg Leu ttc Phe gca Al a 240 gaa G u at c SI e aac Asn at t SIle gga Gy 320 ct g Leu tgc Cys aac Asn ct c Leu aag Lys 400 225 at t l Ie gca Al a cca Pr o gga Gy ctt Leu 305 ttg Leu ggt Gy ttc Phe aag Lys ggc Gy 385 aag Lys ggt Gy gt c Val gcg Al a at t SIle 290 t cc Ser cac Hi s t ac Tyr ggc Gy gcc Al a 370 ggc Gy acc Thr ct g Leu caa G n acc Thr 275 t cc Ser at c SIle tgg Tr p gac Asp ct g Leu 355 at g Met at t SIle t ac Tyr t ac Ty r at g Met 260 gca Al a aac Asn gtt Val cca Pr o ttc Phe 340 gt c Val cgt Ar g t cc Ser ttc Phe tgg Tr p 245 gt g Val ttc Phe ct g Leu at c SIe ct a Leu 325 at t SIe acc Thr cag G n gag G u cgc Ar g 405 787 835 883 931 979 1027 1075 1123 1171 1219 1267 1315 1339 <210> 32 <211> 413 <212> PRT <213> Corynebacter i um gl ut ai cum <400> 32 IVet Ala Ser Lys Leu Thr Thr Thr Ser G n His lie Leu G u Asn Leu 1 5 10 G y G y Pro Asp Asn I e Thr Ser IVet Thr His Cys Ala Thr Arg Leu 25 Arg Phe n Val Lys Asp Gn Ser lie Val Asp On On Ou lie Asp 40 Page 38 seqi i st car r ect ed. t xt Val Val Pro O1n Ov Ser Ser Asp Pr o Ser Val Leu O31n Ly s Ser As p A a Thr 145 Thr Pr o Tr p A a ValI 225 li1e A a Pr o O31y Le u 305 Le u O31y Ph e Ly s O31y 385 ValI As p Ly s A a 115 Le u O31y ValI vb t O31y 1 95 O31y As n Le u O31n Thr 275 Ser I Ie Tr p As p Le u 355 vb t I Ie G31y vb t O31u O31u A a O31n Le u 1 65 O31y A a A a Tyr Tr p 245 ValI Ph e Le u I Ie Le u 325 li1e Thr O31n O31u Ar g 405 O31y 70 Ly s Tyr Ph e Ser As p 150 Hi s A a I Ie O31y Ser 230 ValI Ph e Le u Le u Pr o 310 As n O31n O31y ValI Pr o 390 O31y Ser Hi s O31y Le u Le u 135 Ph e Ser Thr Pr a As p 215 O31y O31u ValI Le u O31u 295 Le u A a O31y ValI Ser 375 Ser Thr 03 y l'bt ValI Ph e O31y Ser 120 li1e Ar g vb t A a A a 200 Thr O31n Ly s Pr a O31y 280 A a Le u I Ie Pr a Ph e 360 Le u Le u A a A a ValI 1 05 As p I Ie A a Tr p A a 1 85 A a ValI ValI O31y Ph e 265 Pr a I Ie Tyr vb t vb t 345 Le u O31y Tyr Tyr O31y O31y Ph e Le u vb t 1 Ser Ly s Le u ValI Pr a 235 Ly s Ser O31y As n Ph e 315 O31n A a Ser vb t ValI 395 O31n A a Tyr Pr a 125 ValI O31u Ph e O31y Pr a 205 O31y Le u I Ie Le u O31y 285 Ser ValI I Ie As n Ly s 365 A a Le u O31u Thr Ser 110 I Ie Le u O31n Tyr A a 1 O31u Le u I Ie I Ie I Ie 270 ValI Pr a Pr a As n Ph e 350 O31u O31y Ar g I Ie O31u Tr p Le u A a Pr a Ph e 1 As n Ph e Pr a A a Pr a 255 vb t O31y Ph e Le u Th r 335 A a Ar g Le u Ph e Lys Thr Tyr Phe Leu Leu Pr o (3y Leu A a A a Page 39 seql i st cor r ect ed. t xt <210> 33 <211> 428 <212> DNA <213> Corynebact eri um gl ut arri cum <220> <221> CDS <222> <223> FRXA01943 <400> 33 cct gac cca atc ttt gca gca Pro Asp Pro lie Phe Ala Ala 1 5 caa cca act gga aac acc gtt G n Pro Thr y Asn Thr Val ctt gtc cag aaa tct gga cac Leu Val G n Lys Ser G y Hi s gtt gaa atc ctt gtc cac gtt Val G u Ile Leu Val His Val 55 gaa ggc ttc acc gtt cac gtt G u y Phe Thr Val His Val 70 gat cca ctg atc act ttt gac Asp Pro Leu lie Thr Phe Asp cct ttg atc acc cca gtt gtg Pro Leu Ile Thr Pro Val Val 100 att gaa ggt att cct gca gat lie G u G y lie Pro Al a Asp 115 aag gtc aac ggc aag aac gag Lys Val Asn Gy Lys Asn G u 130 135 <210> 34 <211> 135 <212> PRT <213> Corynebacteri um gl ut a <400> 34 Pro Asp Pro lie Phe Ala Ala 1 5 G n Pro Thr y Asn Thr Val Leu Val G n Lys Ser G y His Val G u Ile Leu Val His Val 55 ggc G y gtt Val gca Al a 40 gga Gy gag G u gct Al a gt g Val cag G n 120 aag Lys gct Al a gt g Val ttg Leu cgc Ar g gac Asp tct Ser 105 gca Al a ctt Leu 10 cca Pr o gca Al a gac Asp agg Ar g ttc Phe 90 aac Asn aat Asn gga G y gca Al a ttg Leu acc Thr cag G n at t SIle gcc Al a tct Ser cca Pr o gac Asp cgc Ar g gt g Val caa G n cga Ar g gcg Al a t cc Ser ggc G y gct Al a tta Leu caa G n gt c Val t cc Ser aaa Lys acg Thr 125 gca Al a gtc Val agc Ser ggc G y gcg Al a gat Asp ggt G y gtg Val t aacct ggga t ccat gt t gc gca mi cum Sy Lys Leu y Pro y lie Ala lle 10 Val Ala Pro Ala Asp Ala Thr Val le 25 Ala Val Ala Leu Arg Leu Asp Ser y 40 Sy Leu Asp Thr Val G n Leu y y Page INO seqi i st car r ect ed. t xt (3 Gu 1y Phe ThrVal Hi sVal (1u Ar gAr g 1n (1n ValILys Al a 1y 70 75 As p Pro Leu I Ie Thr Phe As p Al a Asp Phe I Ie Ar g Ser Lys Asp Leu 90 SPr o Leu lie Thr Pr o Val Val Val Ser Asn Al a Ala Lys Phe G31y G31u 100 105 110 Ile (1u (1y Ile Pr oAl aAsp (1n Ala Asn Ser Ser Thr Thr Val I Ie 115 120 125 SLys Val Asn (1y Lys Asn (1u 130 135 00 S<210> C1 <211> 18 .C <212> DNA S<213> Art if ici al Sequence <220> <223> Descr i pt i on of Ar t i f i ci al Sequence: pr i rrer <400> ggaaacagt a t gaccat g 18 <210> 36 <211> 17 <212> DNA <213> Art if ici al Sequence <220> <223> Descr i pt i on of Ar t i f i ci al Sequence: pr i rrer <400> 36 gt aaaacgac ggccagt 17 Page 41