AU2007203039A1 - Corynebacterium glutamicum genese encoding proteins involved in carbon metabolism and energy production - Google Patents

Description

P001 Section 29 Regulation 3.2(2) 0\

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Corynebacterium glutamicum genese encoding proteins involved in carbon metabolism and energy production The following statement is a full description of this invention, including the best method of performing it known to us: -1- CORYNEBACTERIUM GLUTAMICUM GENES ENCODING

PROTEINS

INVOLVED IN CARBON METABOLISM AND ENERGY

PRODUCTION

Related Applications This application claims priority to prior U.S. Provisional Patent Application Serial No. 60/141031, filed June 25, 1999, U.S. Provisional Patent Application Serial No. 60/143208, filed July 9, 1999, and U.S. Provisional Patent Application Serial No.

60/151572, filed August 31, 1999. This application also claim's priority to prior German Patent Application No. 19931412.8, filed July 8, 1999, German Patent Application No.

19931413.6, filed July 8, 1999, German Patent Application No. 19931419.5, filed July 8, 1999, German Patent Application No. 19931420.9, filed July 8, 1999, German Patent Application No. 19931424.1, filed July 8, 1999,.German Patent Application No.

19931428.4, filed July 8, 1999, German Patent Application No. 19931431.4, filed July 8, 1999, German Patent Application No. 19931433.0, filed July 8, 1999, German Patent Application No. 19931434.9, filed July 8, 1999, German Patent Application No.

19931510.8, filed July 8, 1999, German Patent Application No. 19931562.0, filed July 8, 1999, German Patent Application No. 19931634.1, filed July 8, 1999, German Patent Application No. 19932180.9, filed July 9, 1999, German Patent Application No.

19932227.9, filed July 9, 1999, German Patent Application No. 19932230.9, filed July 9, 1999, German Patent Application No. 19932924.9, filed July 14, 1999, German Patent Application No. 19932973.7, filed July 14, 1999, German Patent Application No.

19933005.0, filed July 14, 1999, German Patent Application No. 19940765.7, filed August 27, 1999, German Patent Application No. 19942076.9, filed September 3, 1999, German Patent Application No. 19942079.3, filed September 3, 1999, German Patent Application No. 19942086.6, filed September 3, 1999, German Patent Application No.

19942087.4, filed September 3, 1999, German Patent Application No. 19942088.2, filed September 3, 1999, German Patent Application No. 19942095.5, filed September 3, 1999, German Patent Application No. 19942123.4, filed September 3, 1999, and German Patent Application No. 19942125.0, filed September 3, 1999. The entire contents of all of the aforementioned application are hereby expressly incorporated herein by this reference.

-2- 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 the large-scale culture of bacteria developed to produce and secrete large quantities of one or more desired molecules. 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.

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 transformation. These novel nucleic acid molecules encode proteins, referred to herein as sugar metabolism and oxidative phosphorylation (SMP) proteins.

C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The SMP nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, by fermentation processes. Modulation of the expression of the SMP nucleic acids of the invention, or modification of the sequence of the SMP 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 SMP 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 SMP 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 SMP proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetic manipulation of C.

glutamicum and the related Brevibacterium species lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al.,J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals.

This improved production or efficiency of production of a fine chemical may be due to a direct effect of manipulation of a gene of the invention, or it may be due to an indirect effect of such manipulation.

There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glulamicum strain incorporating such an altered protein.

The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH 2 to compounds containing high energy phosphate bonds via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit unfavorable, yet desired metabolic reactions the biosynthesis of a desired fine chemical) to occur.

The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.

(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918; 875-899).

By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor culture. The yield, production, or efficiency of production may be increased, at least r"- O due to the presence of a greater number of viable cells, each producing the N desired fine chemical. Also, many of the degradation products produced during ;sugar metabolism are utilized by the cell as precursors or intermediates in the production of other desirable products, such as fine chemicals. So, by increasing the ability of the cell to metabolize sugars, the number of these degradation products available to the cell for other processes should also be increased.

The invention provides novel nucleic acid molecules which encode 0proteins, referred to herein as SMP proteins, which are capable of, for example, performing a function involved in the metabolism of carbon compounds such as 0 10 sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Nucleic acid molecules encoding an SMP protein are referred to herein as SMP nucleic acid molecules.

In a preferred embodiment, the SMP protein participates in the conversion of carbon molecules and degradation products thereof to energy which is utilized by the cell for metabolic processes. Examples of such proteins include those encoded by the genes set forth in Table 1.

The following embodiments, the subject of this application, are specifically disclosed herein: An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:53, or a complement thereof.

0 An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:54, or a complement thereof.

An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:54, or a complement thereof.

An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:53, or a complement thereof.

An isolated nucleic acid molecule comprising a fragment of at least contiguous nucleotides of the nucleotide sequence of SEQ ID NO:53, or a complement thereof.

An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:54, or a complement thereof.

An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:54.

An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:54.

An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:53.

An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:54.

An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:54, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:54.

An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:53.

A host cell comprising the nucleic acid molecule of SEQ ID NO:53, wherein the nucleic acid molecule is disrupted.

O A host cell comprising the nucleic acid molecule of SEQ ID NO:53, wherein 1 the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:53.

c A host cell comprising the nucleic acid molecule of SEQ ID NO:53, wherein the regulatory region of the nucleic acid molecule is modified relative to the c wild-type regulatory region of the molecule.

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

In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention a sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains an SMP activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. 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 70%, 80%, 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 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 oddnumbered SEQ ID NOs in the Sequence Listing SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein an SMP 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 perform a function involved in the metabolism of carbon compounds such as sugars or the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or has 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 oddnumbered SEQ ID NO in the Sequence Listing) A. Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum SMP protein, or a mC biologically active portion thereof.

Another aspect of the invention pertains to vectors, recombinant expression cN 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 C produce an SMP protein by culturing the host cell in a suitable medium. The SMP 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 an SMP gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by introduction of a nucleic acid molecule of the invention encoding wild-type or mutated SMP sequence as a transgene. In another embodiment, an endogenous SMP gene within the genome of the microorganism has been altered, functionally disrupted, by homologous recombination with an altered SMP gene. In another embodiment, an endogenous or introduced SMP gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SMP protein. In still another embodiment, one or more of the regulatory regions a promoter, repressor, or inducer) of an SMP gene in a microorganism has been altered by deletion, truncation, inversion, or point mutation) such that the expression of the SMP 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 -8sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 782) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.

Still another aspect of the invention pertains to an isolated SMP protein or a portion, a biologically active portion, thereof. In a preferred embodiment, the isolated SMP protein or portion thereof is capable of performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In another preferred embodiment, the isolated SMP protein or portion thereof is sufficiently homologous to an amino acid sequence of the invention a sequence of an even-numbered SEQ ID NO: in the Sequence Listing) such that the protein or portion thereof maintains the ability to perform a function involved in the metabolism of.carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum.

The invention also provides an isolated preparation of an SMP protein. In preferred embodiments, the SMP 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 forth 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 SMP 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 perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or has one or more of the activities set forth in Table 1.

I-9- Alternatively, the isolated SMP protein can comprise an amino acid sequence Swhich is encoded by a nucleotide sequence which hybridizes, hybridizes under ;Z stringent conditions, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%, 97%, or 99% or more homologous to a nucleotide sequence of one of the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred M that the preferred forms of SMP proteins also have one or more of the SMP bioactivities C described herein.

r The SMP polypeptide, or a biologically active portion thereof, can be operatively linked to a non-SMP polypeptide to form a fusion protein. In preferred embodiments, CN this fusion protein has an activity which differs from that of the SMP protein alone. In other preferred embodiments, this fusion protein performs a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates production of a desired compound from the cell.

In another aspect, the invention provides methods for screening molecules which modulate the activity of an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP 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 an SMP 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 an SMP 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 SMP protein activity or SMP nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for one or more C. glutamicum carbon metabolism pathways or for the production of energy through processes such as oxidative phosphorylation, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates SMP protein activity can be an agent which stimulates SMP protein activity or SMP nucleic acid expression. Examples of agents which stimulate SMP protein activity or SMP nucleic acid expression include small molecules, active SMP proteins, and nucleic acids encoding SMP proteins that have been introduced into the cell. Examples of agents which inhibit SMP activity or expression include small molecules and antisense SMP nucleic acid molecules.

Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant SMP 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 be 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 SMP nucleic acid and protein molecules which are involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. The molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms, such as C.

glutamicum, either directly where overexpression or optimization of a glycolytic pathway protein has a direct impact on the yield, production, and/or efficiency of production of, pyruvate from modified C. glutamicum), or may have an indirect -11impact which nonetheless results in an increase of yield, production, and/or efficiency of production of the desired compound where modulation of proteins involved in oxidative phosphorylation results in alterations in the amount of energy available to perform necessary metabolic processes and other cellular functions, such as nucleic acid and protein biosynthesis and transcription/translation). Aspects of the invention are further explicated below.

I. Fine Chemicals The term 'fine chemical' is art-recognized and includes molecules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, agriculture, and cosmetics industries. Such compounds include organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm 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 el 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 art- 12recognized. 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 nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids may be in the D- or L- optical configuration, though Lamino acids are generally the only type found in naturally-occurring proteins.

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

Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetics, agriculture, and pharmaceutical industries. Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine. Glutamate is most commonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine. Glycine, Lmethionine and tryptophan are all utilized in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/ Lmethionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as Nacetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others -13 described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH:

C

i Weinheim, 1985.

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

Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of aketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine c are each subsequently produced from glutamate. The biosynthesis of serine is a three- CI step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both C 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 of pyruvate, the final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate. Isoleucine is formed from threonine. A complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.

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

Thus it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own 14production (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.

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

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

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

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

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

-16- The biosynthesis of vitamin B 12 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also termed 'niacin'. Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by largescale culture of microorganisms, such as riboflavin, Vitamin Br, pantothenate, and biotin. Only Vitamin B 1 2 is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.

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

Several publications have described the use of these chemicals for these medical indications, by influencing purine and/or pyrimidine metabolism Christopherson, R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med Res. Reviews 10: 505-548). Studies of 17enzymes involved in purine and pyrimidine metabolism have been focused on the development of new drugs which can be used, for example, as immunosuppressants or anti-proliferants (Smith, (1995) "Enzymes in nucleotide synthesis." Curr. Opin.

Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates in the biosynthesis of several fine chemicals thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p. 561- 612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide metabolism are increasingly serving as targets against which chemicals for crop protection, including fungicides, herbicides and insecticides, are developed.

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

Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in tum, 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.

18- SD. Trehalose Metabolism and Uses Trehalose consists of two glucose molecules, bound in a, a-1,l linkage. It is c1 commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, Cc cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) U.S.

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

II. Sugar and Carbon Molecule Utilization and Oxidative Phosphorylation Carbon is a critically important element for the formation of all organic compounds, and thus is a nutritional requirement not only for the growth and division of C. glutamicum, but also for the overproduction of fine chemicals from this microorganism. Sugars, such as mono-, di-, or polysaccharides, are particularly good carbon sources, and thus standard growth media typically contain one or more of: glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose (Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim). Alternatively, more complex forms of sugar may be utilized in the media, such as molasses, or other by-products of sugar refinement. Other compounds aside from the sugars may be used as alternate carbon sources, including alcohols ethanol or methanol), alkanes, sugar alcohols, fatty acids, and organic acids acetic acid or lactic acid). For a review of carbon sources and their utilization by microorganisms in culture, see: Ullman's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim; Stoppok, E. and Buchholz, K. (1996) "Sugar-based raw materials for fermentation applications" in Biotechnology (Rehm, H.J. et al., eds.) vol. 6, VCH: Weinheim, p. 5-29; Rehm, H.J.

(1980) Industrielle Mikrobiologie, Springer: Berlin; Bartholomew, and Reiman, H.B. (1979). Economics of Fermentation Processes, in: Peppier, H.J. and Perlman, D., eds. Microbial Technology 2 nd ed., vol. 2, chapter 18, Academic Press: New York; and r7 -19- Kockova-Kratachvilova, A. (1981) Characteristics of Industrial Microorganisms, in: Rehm, H.J. and Reed, eds. Handbook of Biotechnology, vol. 1, chapter 1, Verlag Chemie: Weinheim.

After uptake, these energy-rich carbon molecules must be processed such that they are able to be degraded by one of the major sugar metabolic pathways. Such pathways lead directly to useful degradation products, such as ribose-5-phosphate and phosphoenolpyruvate, which may be subsequently converted to pyruvate. Three of the C most important pathways in bacteria for sugar metabolism include the Embden- N Meyerhoff-Pamas (EMP) pathway (also known as the glycolytic or fructose bisphosphate pathway), the hexosemonophosphate (HMP) pathway (also known as the cN pentose shunt or pentose phosphate pathway), and the Entner-Doudoroff (ED) pathway (for review, see Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York, and Stryer, L. (1988) Biochemistry, Chapters 13-19, Freeman: New York, and references therein).

The EMP pathway converts hexose molecules to pyruvate, and in the process produces 2 molecules of ATP and 2 molecules of NADH. Starting with glucose-1phosphate (which may be either directly taken up from the medium, or alternatively may be generated from glycogen, starch, or cellulose), the glucose molecule is isomerized to fructose-6-phosphate, is phosphorylated, and split into two 3-carbon molecules of glyceraldehyde-3-phosphate. After dehydrogenation, phosphorylation, and successive rearrangements, pyruvate results.

The HMP pathway converts glucose to reducing equivalents, such as NADPH, and produces pentose and tetrose compounds which are necessary as intermediates and precursors in a number of other metabolic pathways. In the HMP pathway, glucose-6phosphate is converted to ribulose-5-phosphate by two successive dehydrogenase reactions (which also release two NADPH molecules), and a carboxylation step.

may also be converted to xyulose-5-phosphate and phosphate; the former can undergo a series of biochemical steps to glucose-6-phosphate, which may enter the EMP pathway, while the latter is commonly utilized as an intermediate in other biosynthetic pathways within the cell.

The ED pathway begins with the compound glucose or gluconate, which is subsequently phosphorylated and dehydrated to form 2-dehydro-3-deoxy-6-P-gluconate.

Glucuronate and galacturonate may also be converted to 2-dehydro-3-deoxy-6-Pgluconate through more complex biochemical pathways. This product molecule is subsequently cleaved into glyceraldehyde-3-P and pyruvate; glyceraldehyde-3-P may itself also be converted to pyruvate.

The EMP and HMP pathways share many features, including intermediates and enzymes. The EMP pathway provides the greatest amount of ATP, but it does not produce ribose-5-phosphate, an important precursor for, nucleic acid biosynthesis, nor does it produce erythrose-4-phosphate, which is important for amino acid biosynthesis. Microorganisms that are capable of using only the EMP pathway for glucose utilization are thus not able to grow on simple media with glucose as the sole carbon source. They are referred to as fastidious organisms, and their growth requires inputs of complex organic compounds, such as those found in yeast extract.

In contrast, the HMP pathway produces all of the precursors necessary for both nucleic acid and amino acid biosynthesis, yet yields only half the amount of ATP energy that the EMP pathway does. The HMP pathway also produces NADPH, which may be used for redox reactions in biosynthetic pathways. The HMP pathway does not directly produce pyruvate, however, and thus these microorganisms must also possess this portion of the EMP pathway. It is therefore not surprising that a number of microorganisms, particularly the facultative anerobes, have evolved such that they possess both of these pathways.

The ED pathway has thus far has only been found in bacteria. Although this pathway is linked partly to the HMP pathway in the reverse direction for precursor formation, the ED pathway directly forms pyruvate by the aldolase cleavage of 3ketodeoxy-6-phosphogluconate. The ED pathway can exist on its own and is utilized by the majority of strictly aerobic microorganisms. The net result is similar to that of the HMP pathway, although one mole of ATP can be formed only if the carbon atoms are converted into pyruvate, instead of into precursor molecules.

The pyruvate molecules produced through any of these pathways can be readily converted into energy via the Krebs cycle (also known as the citric acid cycle, the citrate cycle, or the tricarboxylic acid cycle (TCA cycle)). In this process, pyruvate is first decarboxylated, resulting in the production of one molecule of NADH, 1 molecule of acetyl-CoA, and 1 molecule of CO 2 The acetyl group of acetyl CoA then reacts with -21 the 4 carbon unit, oxaolacetate, leading to the formation of citric acid, a 6 carbon organic acid. Dehydration and two additional CO 2 molecules are released. Ultimately, Soxaloacetate is regenerated and can serve again as an acetyl acceptor, thus completing the cycle. The electrons released during the oxidation of intermediates in the TCA cycle are transferred to NAD to yield NADH.

During respiration, the electrons from NADH are transferred to molecular m oxygen or other terminal electron acceptors. This process is catalyzed by the respiratory chain, an electron transport system containing both integral membrane proteins and C membrane associated proteins. This system serves two basic functions: first, to accept electrons from an electron donor and to transfer them to an electron acceptor, and N second, to conserve some of the energy released during electron transfer by the synthesis of ATP. Several types of oxidation-reduction enzymes and electron transport proteins are known to be involved in such processes, including the NADH dehydrogenases, flavin-containing electron carriers, iron sulfur proteins, and cytochromes. The NADH dehydrogenases are located at the cytoplasmic surface of the plasma membrane, and transfer hydrogen atoms from NADH to flavoproteins, in turn accepting electrons from NADH. The flavoproteins are a group of electron carriers possessing a flavin prosthetic group which is alternately reduced and oxidized as it accepts and transfers electrons.

Three flavins are known to participate in these reactions: riboflavin, flavin-adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). Iron sulfur proteins contain a cluster of iron and sulfur atoms which are not bonded to a heme group, but which still are able to participate in dehydration and rehydration reactions. Succinate dehydrogenase and aconitase are exemplary iron-sulfur proteins; their iron-sulfur complexes serve to accept and transfer electrons as part of the overall electron-transport chain. The cytochromes are proteins containing an iron porphyrin ring (heme). There are a number of different classes of cytochromes, differing in their reduction potentials.

Functionally, these cytochromes form pathways in which electrons may be transferred to other cytochromes having increasingly more positive reduction potentials. A further class of non-protein electron carriers is known: the lipid-soluble quinones coenzyme These molecules also serve as hydrogen atom acceptors and electron donors.

-22- The action of the respiratory chain generates a proton gradient across the cell membrane, resulting in proton motive force. This force is utilized by the cell to synthesize ATP, via the membrane-spanning enzyme, ATP synthase. This enzyme is a multiprotein complex in which the transport of H' molecules through the membrane results in the physical rotation of the intracellular subunits and concomitant phosphorylation of ADP to form ATP (for review, see Fillingame, R.H. and Divall, S.

(1999) Novartis Found. Symp. 221: 218-229, 229-234).

Non-hexose carbon substrates may also serve as carbon and energy sources for cells. Such substrates may first be converted to hexose sugars in the gluconeogenesis pathway, where glucose is first synthesized by the cell and then is degraded to produce energy. The starting material for this reaction is phosphoenolpyruvate (PEP), which is one of the key intermediates in the glycolytic pathway. PEP may be formed from substrates other than sugars, such as acetic acid, or by decarboxylation of oxaloacetate (itself an intermediate in the TCA cycle). By reversing the glycolytic pathway (utilizing a cascade of enzymes different than those of the original glycolysis pathway), glucose-6phosphate may be formed. The conversion ofpyruvate to glucose requires the utilization of 6 high energy phosphate bonds, whereas glycolysis only produces 2 ATP in the conversion of glucose to pyruvate. However, the complete oxidation of glucose (glycolysis, conversion of pyruvate into acetyl CoA, citric acid cycle, and oxidative phosphorylation) yields between 36-38 ATP, so the net loss of high energy phosphate bonds experienced during gluconeogenesis is offset by the overall greater gain in such high-energy molecules produced by the oxidation of glucose.

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 SMP nucleic acid and protein molecules, which participate in the conversion of sugars to useful degradation products and energy ATP) in C. glutamicum or which may participate in the production of useful energy-rich molecules ATP) by other processes, such as oxidative phosphorylation. In one embodiment, the SMP molecules participate in the metabolism of carbon compounds such as sugars or the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In a preferred embodiment, -23 the activity of the SMP molecules of the present invention to contribute to carbon metabolism or energy production in C. glutamicum has an impact on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the SMP molecules of the invention are modulated in activity, such that the C. glutamicum metabolic and energetic pathways in which the SMP proteins of the invention participate are modulated in yield, production, and/or efficiency of production, which either directly or indirectly modulates the yield, production, and/or efficiency of production of a desired fine chemical by C. glutamicum.

The language, "SMP protein" or "SMP polypeptide" includes proteins which are capable of performing a function involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Examples of SMP proteins include those encoded by the SMP genes set forth in Table 1 and by the odd-numbered SEQ ID NOs. The terms "SMP gene" or "SMP nucleic acid sequence" include nucleic acid sequences encoding an SMP protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions. Examples of SMP 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 -24products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process. The term "degradation product" is artrecognized and includes breakdown products of a compound. Such products may themselves have utility as precursor (starting point) or intermediate molecules necessary for the biosynthesis of other compounds by the cell. The language "metabolism" is artrecognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, then, the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.

In another embodiment, the SMP molecules of the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incorporating such an altered protein. The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH 2 to more useful forms via oxidative phosphdrylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit unfavorable, yet desired metabolic reactions the biosynthesis of a desired fine chemical) to occur.

The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.

g (1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918; 875-899).

By improving the growth and multiplication of these engineered cells, it is possible to c1 increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor CN culture. The yield, production, or efficiency of production may be increased, at least due to the presence of a greater number of viable cells, each producing the desired fine chemical. Further, a number of the degradation and intermediate compounds produced during sugar metabolism are necessary precursors and intermediates for other biosynthetic pathways throughout the cell. For example, many amino acids are synthesized directly from compounds normally resulting from glycolysis or the TCA cycle serine is synthesized from 3-phosphoglycerate, an intermediate in glycolysis). Thus, by increasing the efficiency of conversion of sugars to useful energy molecules, it is also possible to increase the amount of useful degradation products as well.

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

glutamicum SMP proteins are shown in the Sequence Listing as odd-numbered SEQ ID NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode proteins having a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum.

The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention -26the 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.

An SMP protein or a biologically active portion or fragment thereof of the invention can participate in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or can 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 SMP 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 SMP-encoding nucleic acid SMP 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 of sequence upstream from the 5' end of the coding region and at least about nucleotides of sequence downstream from the 3'end of the coding region of the gene.

The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid sequences located at the 5' and 3' ends of the nucleic acid) in the -27genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated SMP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide 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 SMP 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 a'll or a portion of one of the nucleic acid sequences of the invention an odd-numbered SEQ ID NO:) 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-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can be prepared using reverse transcriptase Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Sequence Listing. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and -28appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an SMP 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 SMP DNAs of the invention. This DNA comprises sequences encoding SMP 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 sequences in 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, or RXS number having the designation "RXA," "RXN," or "RXS" followed by digits RXA01626, RXN00043, or RXS0735). 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, or RXS 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 also be distinguished by their differing RXA, RXN, or RXS designations. The coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also set forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence. For example, the coding region for RXA02735 is set forth in SEQ ID NO:1, while the amino acid sequence which it encodes is set forth as SEQ ID NO:2. The sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, or RXS designations as the amino acid molecules which they encode, such that they can be readily correlated. For example, the amino acid sequence designated -29- RXA00042 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXA00042, and the amino acid sequence designated RXN00043 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXN00043. The correspondence between the RXA, RXN and RXS nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs is set forth in Table 1.

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 RXAdesignation. For example, SEQ ID NO:11, designated, as indicated on Table 1, as "F RXA01312", is an F-designated gene, as are SEQ ID NOs: 29, 33, and 39 (designated on Table I as "F RXA02803", "F RXA02854", and "F RXA01365", respectively).

In one embodiment, the nucleic acid molecules of the present invention are not intended to include 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 Listing the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.

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

to be included. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention, 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 an SMP protein. The nucleotide sequences determined from the cloning of the SMP genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning SMP homologues in other cell types and organisms, as well as SMP 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 odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone SMP homologues. Probes based on the SMP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g.

the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells which misexpress an SMP protein, such as by measuring a level of an SMP-encoding -31 nucleic acid in a sample of cells, detecting SMP mRNA levels or determining whether a genomic SMP 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 perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. 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 even-numbered 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 able to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum.

Protein members of such sugar metabolic pathways or energy producing systems, as described herein, may play a role in the production and secretion of one or more fine chemicals. Examples of such activities are also described herein. Thus, "the function of an SMP protein" contributes either directly or indirectly to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of SMP protein activities are set forth in Table 1.

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

Portions of proteins encoded by the SMP nucleic acid molecules of the invention are preferably biologically active portions of one of the SMP proteins. As used herein, the term "biologically active portion of an SMP protein" is intended to include a portion, a domain/motif, of an SMP protein that participates in the metabolism of carbon -32compounds such as sugars, or in energy-generating pathways in C. glutamicum, or has an activity as set forth in Table 1. To determine whether an SMP protein or a biologically active portion thereof can participate in the metabolism of carbon compounds or in the production of energy-rich molecules in C. glutamicum, 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 an SMP 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 SMP protein or peptide by recombinant expression in vitro) and assessing the activity of the encoded portion of the SMP 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 SMP 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.

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

It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to the present invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art a Genbank sequence (or the protein encoded by such a sequence) set forth in Tables 2 or For example, the invention includes a nucleotide sequence which is greater than and/or at least 58% identical to the nucleotide sequence designated RXA00014 (SEQ ID NO:41), -33 a nucleotide sequence which is greater than and/or at least identical to the nucleotide sequence designated RXA00195 (SEQ ID NO:399), and a nucleotide sequence which is greater than and/or at least 42% identical to the nucleotide sequence designated RXAOO 196 (SEQ ID NO:401). 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 SMP 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 SMP proteins may exist within a population the C.

glutamicum population). Such genetic polymorphism in the SMP gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an SMP protein, preferably a C. glutamicum SMP protein. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the SMP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in SMP that are the result of natural variation and that do not alter the functional activity of SMP proteins are intended to be within the scope of the invention.

Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. gluramicum SMP DNA of the invention can be isolated based on their homology to the C. glutamicum SMP 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 -34 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 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 75% 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 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 45°C, 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 encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C.

glutamicum SMP protein.

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

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding SMP proteins that contain changes in amino acid residues that are not essential for SMP activity. Such SMP 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 SMP 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 participate in the metabolism of carbon compounds such as sugars, or in the biosynthesis of high-energy compounds in C. glutamicum, 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 ID NOs of the Sequence Listing, more preferably at least about 70% homologous to one of these sequences, even more preferably at least about 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.

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

-36- An isolated nucleic acid molecule encoding an SMP 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 an SMP protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an SMP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an SMP activity described herein to identify mutants that retain SMP activity. Following mutagenesis of the nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).

In addition to the nucleic acid molecules encoding SMP proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded DNA molecule or -37complementary 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 SMP 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 an SMP 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 NO. 3 (RXA01626) comprises nucleotides 1 to 345). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding SMP. 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 SMP 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 SMP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of SMP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SMP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2 thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- -38galactosylqueosine, 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 an SMP protein to thereby inhibit expression of the protein, by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred.

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

Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- -39 methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave SMP mRNA transcripts to thereby inhibit translation of SMP mRNA. A ribozyme having specificity for an SMP-encoding nucleic acid can be designed based upon the nucleotide sequence of an SMP cDNA disclosed herein SEQ ID NO. 3 (RXA01626)). 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 an SMP-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, SMP 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, SMP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an SMP nucleotide sequence an SMP promoter and/or enhancers) to form triple helical structures that prevent transcription of an SMP gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27- 36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

B. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an SMP protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form 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 host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells.

Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, lacI q T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SP02, 1-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, CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, -41 usp, STLS 1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by those 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 SMP proteins, mutant forms of SMP proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for expression of SMP proteins in prokaryotic or eukaryotic cells. For example, SMP 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. el 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 tumefaciens -mediated transformation ofArabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep: 583-586), or mammalian cells.

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

Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion -42expression 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 pRITS (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 SMP 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 SMP 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, pUCI9, pKC30, pRep4, pHS pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- III 113-B 1, .gtl 1, pBdCl, and pET 1 Id (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 transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 d vector relies on transcription from a T7 gnl0-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS 174(DE3) from a resident X prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJ 101, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUBl 10, pC194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBLI, pSA77, or pAJ667 (Pouwels et al., eds.

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

-43 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 SMP protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), 2 pAG-1, Yep6, Yep 3, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C.A.M.J.J. Punt, P.J.

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

Alternatively, the SMP 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 Sf9 cells) include the pAc series (Smith et al.

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

In another embodiment, the SMP proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, Kemper, Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721, and include pLGV23, pGHlac+, -44pBIN19, 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 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; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

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

The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews Trends in Genetics, Vol. 1(1) 1986.

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

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

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" 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, -46transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, 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 an SMP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, 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 an SMP gene into which a deletion, addition or substitution has been introduced to thereby alter, functionally disrupt, the SMP gene.

Preferably, this SMP gene is a Corynebacterium glutamicum SMP gene, but it can be a 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 SMP 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 SMP gene is mutated or otherwise altered but still encodes functional protein the upstream regulatory region can be altered to thereby alter the expression of the endogenous SMP protein). In the homologous recombination vector, the altered portion of the SMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the SMP gene to allow for homologous recombination to occur between the exogenous SMP gene carried by the vector and an endogenous SMP gene in a microorganism. The additional -47flanking SMP 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 SMP gene has homologously recombined with the endogenous SMP 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 an SMP gene on a vector placing it under control of the lac operon permits expression of the SMP gene only in the presence of IPTG. Such regulatory systems are well known in the art.

In another embodiment, an endogenous SMP 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 SMP gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SMP protein. In still another embodiment, one or more of the regulatory regions a promoter, repressor, or inducer) of an SMP gene in a microorganism has been altered by deletion, truncation, inversion, or point mutation) such that the expression of the SMP gene is modulated. One of ordinary skill in the art will appreciate that host cells containing more than one of the described SMP 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) an SMP protein. Accordingly, the invention further provides methods for producing SMP proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an SMP protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered SMP protein) in a suitable medium until SMP protein is produced. In another -48embodiment, the method further comprises isolating SMP proteins from the medium or the host cell.

C. Isolated SMP Proteins Another aspect of the invention pertains to isolated SMP 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 SMP 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 SMP protein having less than about 30% (by dry weight) of non-SMP protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-SMP protein, still more preferably less than about 10% of non-SMP protein, and most preferably less than about 5% non-SMP protein. When the SMP 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 SMP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of SMP protein having less than about 30% (by dry weight) of chemical precursors or non-SMP chemicals, more preferably less than about 20% chemical precursors or non-SMP chemicals, still more preferably less than about 10% chemical precursors or non-SMP chemicals, and most preferably less than about 5% chemical precursors or non-SMP chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the SMP protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum SMP protein in a microorganism such as C. glutamicum.

-49- O An isolated SMP protein or a portion thereof of the invention can participate in the metabolism of carbon compounds such as sugars, or in the production of energy ;Z compounds by oxidative phosphorylation) utilized to drive unfavorable metabolic pathways, 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 r n thereof maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. The portion of the protein ri is preferably a biologically active portion as described herein. In another preferred embodiment, an SMP 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 SMP 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 SMP protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 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 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 SMP proteins of the present invention also preferably possess at least one of the SMP activities described herein. For example, a preferred SMP protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or which has one or more of the activities set forth in Table 1.

In other embodiments, the SMP protein is substantially homologous to an amino acid sequence of 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 SMP 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 SMP 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.

Biologically active portions of an SMP protein include peptides comprising amino acid sequences derived from the amino acid sequence of an SMP protein, an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to an SMP protein, which include fewer amino acids than a full length SMP protein or the full length protein which is homologous to an SMP protein, and exhibit at least one activity of an SMP protein.

Typically, biologically active portions (peptides, peptides which are, for example, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of an SMP protein. Moreover, other -51 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 an SMP protein include one or more selected domains/motifs or portions thereof having biological activity.

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

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

-52- Preferably, an SMP 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, Ausubel et al., eds. John Wiley Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety a GST polypeptide).

An SMP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-franie to the SMP protein.

Homologues of the SMP protein can be generated by mutagenesis, discrete point mutation or truncation of the SMP protein. As used herein, the term "homologue" refers to a variant form of the SMP protein which acts as an agonist or antagonist of the activity of the SMP protein. An agonist of the SMP protein can retain substantially the same, or a subset, of the biological activities of the SMP protein. An antagonist of the SMP protein can inhibit one or more of the activities of the naturally occurring form of the SMP protein, by, for example, competitively binding to a downstream or upstream member of the sugar molecule metabolic cascade or the energy-producing pathway which includes the SMP protein.

In an alternative embodiment, homologues of the SMP protein can be identified by screening combinatorial libraries of mutants, truncation mutants, of the SMP protein for SMP protein agonist or antagonist activity. In one embodiment, a variegated library of SMP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of SMP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SMP 53 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins for phage display) containing the set of SMP sequences therein.

There are a variety of methods which can be used to produce libraries of potential SMP 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 SMP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, Narang, S.A. (1983) Tetrahedron 39:3; Itakura el 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 SMP protein coding can be used to generate a variegated population of SMP fragments for screening and subsequent selection of homologues of an SMP protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an SMP 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 S1 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 SMP protein.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of SMP homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the -54frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify SMP 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 SMP library, using methods well known in the art.

O

D. Uses and Methods of the Invention C1 The nucleic acid molecules, proteins, protein homologues, fusion proteins, Sprimers, vectors, and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms; mapping of genomes of organisms related to C. glutamicum; identification and localization of C.

glutamicum sequences of interest; evolutionary studies; determination of SMP protein regions required for function; modulation of an SMP protein activity; modulation of the metabolism of one or more sugars; modulation of high-energy molecule production in a cell ATP, NADPH); and modulation of cellular production of a desired compound, such as a fine chemical.

The SMP 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; byprobing 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 pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology. In this disease, a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells; the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease. Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.

In one embodiment, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium 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 of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutamicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of C. glutamicum, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.

The SMP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and energy-releasing processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the -56evolutionary 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 SMP nucleic acid molecules of the invention may result in the production of SMP proteins having functional differences from the wild-type SMP 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 an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more SMP 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 SMP protein is assessed.

There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incorporating such an altered protein.

The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH 2 to more useful forms via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit -57unfavorable, yet desired metabolic reactions the biosynthesis of a desired fine chemical) to occur.

Further, modulation of one or more pathways involved in sugar utilization permits optimization of the conversion of the energy contained within the sugar molecule to the production of one or more desired fine chemicals. For example, by reducing the activity of enzymes involved in, for example, gluconeogenesis, more ATP is available to drive desired biochemical reactions (such as fine chemical biosyntheses) in the cell. Also, the overall production of energy molecules from sugars may be modulated to ensure that the cell maximizes its energy production from each sugar molecule. Inefficient sugar utilization can lead to excess CO 2 production and excess energy, which may result in futile metabolic cycles. By improving the metabolism of sugar molecules, the cell should be able to function more efficiently, with a need for fewer carbon molecules. This should result in an improved fine chemical product: sugar molecule ratio (improved carbon yield), and permits a decrease in the amount of sugars that must be added to the medium in large-scale fermentor culture of such engineered C.

glutamicum.

The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler el al.

(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918; 875-899).

By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor culture. The yield, production, or efficiency of production may be increased, at least -58 due to the presence of a greater number of viable cells, each producing the desired fine chemical.

Further, many of the degradation products produced during sugar metabolism are themselves utilized by the cell as precursors or intermediates for the production of a number of other useful compounds, some of which are fine chemicals. For example, pyruvate is converted into the amino acid alanine, and ribose-5-phosphate is an integral part of, for example, nucleotide molecules. The amount and efficiency of sugar metabolism, then, has a profound effect on the availability of these degradation products in the cell. By increasing the ability of the cell to process sugars, either in terms of efficiency of existing pathways by engineering enzymes involved in these pathways such that they are optimized in activity), or by increasing the availability of the enzymes involved in such pathways by increasing the number of these enzymes present in the cell), it is possible to also increase the availability of these degradation products in the cell, which should in turn increase the production of many different other desirable compounds in the cell fine chemicals).

The aforementioned mutagenesis strategies for SMP proteins to result in increased yields of a fine chemical from C. glutamicum are not meant to be limiting; variations on these strategies will be readily apparent to one of ordinary skill in the art.

Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and protein molecules of the invention may be utilized to generate C. glulamicum or related strains of bacteria expressing mutated SMP 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 product produced by C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C. glutamicum strain of the invention.

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

2007203039 29 Jun 2007 TABLE 1: GENES IN THE APPLICATION

HMP:

Nucleic Acid Amino Acid SEQ ID NO SEQ 10 NO 1 2 3 4 6 7 8

TCA:

Nucleic Acid SEQ ID NO 9 11 13 17 19 21 Amino Acid SEQ ID NO 10 12 14 16 18 20 22 Identification Code RXS02735 RXA0 1626 RXA02245 RXAOI 015 Identification Code R.XNO 1312 F RXAOI1312 RXN00231 RXAO 131 1 RXAO1 535 RXA00517 RXA01 350 GontiR WV0074 GR00452 GRO0654 GR00290 Conti W0082 GR00380 VV0083 GR00380 GR00427 GRO01 31 GR00392 '14576 4270 13639 346 15280 3926 14295 5 NT Start NT Stop Function 6-Phosphoglucolactonase L-ribulose-phosphae 4-epimerase RIBULOSE-PHOSPHATE 3-EPIMERASE (EC 5.1.3.1) RIBOSE 5-PHOSPHATE ISOMERASE (EC 5.3.1.6) NT Start NT Stop Function 20803 2690 15484 1611 1354 1407 1844 18785 1614 14015 865 2760 2447 2827 SUCCINATE DEHYDROGENASE FLAVOPROTEIN SUBUNIT (EC 1. 3. 99. 1) SUCCINATE DEHYDROGENASE FLAVOPROTEIN SUBUNIT (EC 11.3:91.1) SUCCINATE-SEMIALDEHYDE DEHYOROGENASE (NADP+) (EC 1.2.1.16) SUCCINATE DEHYDROGENASE IRON'-SULFUR PROTEIN (EC 1.3.99.1) FUMARATE HYDRATASE PRECURSOR (EC 4.2.1.2) MALATE DEHYOROGENASE (EC 1.1.1.37) (EC 1.1.1.82) MALATE DEHYDROGENASE (EC 1. 1. 1.37) EMB-Pathway Nucleic Amino Acid Acid SEQ SEQ ID NO ID NO 23 24 26 27 28 29 30 31 32 33 34 36 Identification Code RXA02 149 RXA0 1814 RXN02803 F RXA02803 RXN03076 F RXA02854 R.X(AOO51 1 Coni GR00639 GROO515 W0086 GR00784 W0043 GRI 0002 GROO 129 NT Start NT Stop Function 17786 2571 2 1624 1588 18754 910 657 400 35 5 513 GLUCOKINASE (EC 2.7.1.2) PHOSPHOGLUCOMUTASE (EC 5.4.2.2)1/ PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2)1/ PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (C 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2)1/ PI-OSPHOMANNOMUTASE (EC 5.4.2.8) 2007203039 29 Jun 2007 Table I (continued) Nucleic Acid Amino Acid Identification Code Conli NT Start NT Stop Function SEQ ID NO SEQ 10 NO 37 38 RXN01365 WV0091 1476 103 PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) 39 40 F RXA0 1365 GR00397 897 4 PHOSPtIOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOWLANNOMUTASE (C 5.4.2.8) 41 42 RXA00098 GROO014 6525 8144 GLUCOSE -6-PHOSPHATE ISOMERASE (GPI) (EC 5.3.1.9) 43 44 RXA01989 GR00578 1 630 GLUCOSE-6-PHOSPHATE ISOMERASE A (GPI A) (EC 5.3.1.9) 46 RXAOO340 GR00059 1549 2694 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 47 48 RXA02492 GR00720 2201 2917 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 49 50 RYA60381 GR00082 1451 846 PH-OSPHOGLYCERATE MUTASE (C 5.4.2.1) 51 52 RXA02122 GR00636 6511 5813 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.11) 53 54 RXAOO206 GR00032 6171 5134 6-PHOSPHOFRUCTOKtNASE (EC 2.7.1.11) 56 RXA01243 GR00359 2302 3261 1-PHOSPHOFRUCTOKINASE (C 2.7.1.56) 57 58 RXA01882 GR00538 1165 2154 1-PHOSPHOFRUCTOKtNASE (EC 2.7.1.56) 59 60 RXAO 1702 GR00479 1397 366 FRUCTOSE-BISPHOSPHATE ALOOLASE (EC 4.1.2.13) 61 62 RXA02258 GR00654 26451 27227 TRIOSEPHOSPHATE ISOMERASE (EC 5.3.1.1) 63 64 RXN0I 225 WV0064 6382 4943 GLYCERALIDEHYDE 3-PHOSPHATE DEHYDROGENASE (CO 1.2.1.12) 66 F RXA01 225 GR00354 5302 6741 GLYCERALIDEHYDE-3-PHOSPHATE DEHYDROGENASE HOMOLOG 67 68 RXA02256 GR00654 23934 24935 GLYCERALODEHYDE 3-PHOSPHATE DEHYDROGENASE (EC 1.2.1.12) 69 70 RXA02257 GR00654 25155 26369 PHOSPHOGLYCE RATE KINASE (EC 2.7.2.3) 71 72 RXA00235 GR00036 2365 1091 ENOLASE (EC 4.2.1.11) 73 74 RXA01093 GR00306 1552 122 PYRUVATE KINASE (EC 2.7.1.40) 76 RXN02675 WV0098 72801 70945 PYRUVATE KINASE (EC 2.7.1.40) 77 78 F RXA02675 G1300754 2 364 PYRUVATE KINASE (EC 2.7.1.40) a 79 80 F RXA02695 GR00755 2949 4370 P YRUVATE KINASE (EC 2.7.1.40) 81 82 RXA00682 GR00179 5299 3401 PHOSPHOENOLPYRUVATE SYNTH-ASE (EC 2.7.9.2) 83 84 R.XA00663 GRO0179 6440 53.49 PHOSPHOENOLPYRUVATE SYNTHASE (C 2.7.9.2) 86 RXN00635 WV0135 22708 20972 PYRUVATE DEHYDROGENASE (CYTOCHROME) (C 1.2.2.2) 87 88 F RXA02807 GR00788 86 552 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC 1.2.2.2) 89 90 F RXA00635 GRO0167 3 923 PYRUVATE DEHYDROGENASE (CYTOCHROME) (C 1.2.2.2) 91 92 RXN03044 W00b19 1391 2221 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 93 94 F RXA02852 GR00852 3 281 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 96 F RXA00268 GROO041 125 955 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 97 98 RXN03086 WV0049 2243 2650 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 99 100 F RXA02887 GR10022 411 4 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 101 102 RXN03043 WOO 19 1 1362 PYRUVATE DEI-YDROGENASE El COMPONENT (EC 1.2.4.1) 103 104 F RXA02897 GR10039 1291 5 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 105 106 RXN03083 WV0047 88 '1110 DIHYOROLIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 107 108 F RXA02853 GRIO001 89 1495 DIHYDROLIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 1109 110 RXA02259 GR00654 27401 30172 PHOSPHOENOLPYRUVATE CARBOXYLASE (EC 4.1.1.31) ill 112 RXN02326 WV0047 4500 5315 PYRUVATE CARBOXYLASE (C 6.4. 1.1) 113 114 F RXA02326 GR00668 5338 4523 PYRUVATE CARBOXYLASE 115 116 RXN02327 WV0047 3533 4492 PYRUVATE CARBOXYLASE EC 6.4. 1.1) 117 118 F RXA02327 GRG0668 6305 53.46 PYRUVATE CARBOXYLASE 119 120 RXN02328 WV0047 1842 3437 PYRUVATE CARBOXYLASE (EC 6.4. 1. 1) 121 122 F RXA02328 GR00668 7783 6401 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 123 124 RXN01048 WV0079 12539 11316 MALIC ENZYME (EC 1.1.1.39) 2007203039 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 125 127 1 29 131 133 135 137 139 141 143 145 147 149 151 153 155 157 159 161 163 165 167 169 171 173 175 177 179 181 Amino Acid SEQ I0 NO 126 128 130 132 134 136 138 140 142 144 146 148 150 152 154 156 Identific-ation Code F RXA01048 F RXA00290 RXA02694 RXN00296 F RXA00296 RXA0 1901 RXN01 952 F RXA01952 F R.XA01 955 RXA00293 RXN01 130 F RXA01I130 RXN031 12 F RXAO1 133 RXN00871 F RXA00871 Con GR00296 GR00046 GR00755 W0O176 GR00048 GR00544 WVO 105 GR00562 GR00562 GR00047 WVO 157 GR00315 VV0085 GR00316 VVO 127 GR00239 W0354 GR00816 W00O19 GR00422 GROO211 W0213 GR00690 VV0098 W0052 W0377 W0382 W0098 W0009 W0009 3.

4693 1879 35763 3 4158 9954 4611 2645 6138 2 509 568 3127 23.44 287 287 7474 1250 3993 6135 1390 59053 3216 310 3 14370 3477 3703 290 5655 2820 38606 2837 5417 11666 216 6209 1734 5536 304 6 1116 2240 3207 559 562 8298 2074 2989 5224 686 58385 3428 519 281 12541 2296 3533 MALIC ENZYME (EC 1.1.1.39) MALIC ENZYME (EC 1.1.1.39) L-LACTATE DEHYDROGENASE (EC 1.1.1,27) 0-LACTATE DEHYDROGENASE (CYTOCHROME) (EC 1.1.2.4) 0-LACTATE DEHYDROGENASE (CYTOCHROME) (EC 1.1.2.4) 1-LACTATE 0EHYDROGENASE (CYTOCHROME) (EC 1.1.2.3) 0-LACTATE DEH-YDROGENASE (EC 1.1.1.28) 0-LACTATE DEHYOROGENASE (EC 1.1.1.28) 0-LACTATE DEHYDROGENASE (EC 1.1.1.28) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) 0-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) D-3-PHOSPHOGLYCERATE DEHYOROGENASiE (EC 1.1.1.95) D-3-PHOSPHOGLYCtERAft DEHYDROGENA*SE (EC 1.1. 1.95) D-3-PHOSPH-OGLYCERATE DEHYObROGENASE (EC 1.1.1.95) IQIB PROTEIN I018 PROTEIN: 0-FRUCTOSE 1 .6-B3ISPHOSPHATE GLYCERONE-CC PHO0SPHATE D- GLYCERALDEHYDE 3-PHOSPHATE.

lOLS PROTEIN lOLS PROTEIN NAGO PROTEIN PUTATIVE N-GLYCERALDEHYDE-2-PHOSPHOTPANSFERASE GLPX PROTEIN D-3-PHOSPHOGLYCERATE DEHYOROGENASE (EC 1.1.1.95) D-3-PHOSPHOOLYCERATE DEHYDROGENASE (EC 1.1.1.95) PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) PYRUVATE CARBOXYLASE (EC 6.4.1.1) PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) PHOSPHOENOLPYRUVATE CARBOXYKINASE (GTPJ (EC 4.1.1.32) LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED- CHAIN AIPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4) LIPOAMIIJE DEHYDROGENASE COMPONENT (E3) OF BRANCHED- CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4) RXN02829 F RXA02829 RXN0 1468 F RXA01468 RXA00794 RXN02920 F RXA02379 RXN02688 RXN03087 RXN03 186 RXN031 87 RXN02591 RXS0 1260 RXS01 261 183 184 Glycerol metabolism Nucleic Acid SEQ ID NO 185 187 189 191 193 195 Amino Acid Identification Code SEQ ID NO 186 RXA02640 188 RXN01025 '190 F RXA01025 192 RXA01851 194 RXA01242 196 RXA02288 C Onlg GR00749 WO0143 GR00293 GR00525 GR00359 GR00661 NT Start NT Stop Function 1400 5483 939 3515 1526 992 GLYCEROL KINASE (EC 2.7.1.30) GLYCEROL-3-PHOSPHATE DEHYOROGENASE (EC 1. 1. 1.94) GLYCEROL.3-PHOSPHATE DEHYDROGENASE (EC 1.1.1.94) AEROBIC GLYCEROL-3-PHOSPHATE DEHYDROGENASE (EC 1. 1.99.5) GLYCEROL-3-PHOSPHATE REGULON REPRESSOR GLYCEROL-3-PHOSPHATE REGULON REPRESSOR 2007203039 29 Jun 2007 Table I (continued) NT Start NT Stop Function Nucleic Acid Amino Acid Identification Code SEQ ID NO SEQ ID NO 197 198 RXN01891 Cog.

199 200 201 202 203 204 F RXA01891 RXA02414 RXN01 580 WV0122 24949 24086 GLYCEROL-3-PHOSPHATE-BINDING PERIPLASMIC PROTEIN

PRECURSOR

GR00541 1736 918 GLYCEROL-3-PHOSPHATE- BINDING PERIPLASMIC PROTEIN

PRECURSOR

CR00703 3808 3062 Uncharacterized protein involved in glycerol metabolism (homolog of Drosophila rhomboid) WV0122 22091 22807 Clycerophosphoryl diester phosphodiesterase Acetate metabolism Nucleic Acid Amino Acid Identification Code Cti. NT Start NT Stop Function SEQ ID NO SEQ ID NO 205 206 RXA01436 GR00418 2547 1357 ACETATE KINASE (EC: 2.7.2.1) 207 208 RXA00686 GR00179 8744 7941 ACETATE OPERON REPRESSOR 209 210 RXA00246 CR00037 4425 3391 ALCOHOL DEHYOROGENASE (EC 1.1.1.1) 211 212 RXAO 1571 CR00438 1360 1959 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) 213 214 RXA01572 CR00438 1928 2419 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) 215 216 RXA01758 CR00498 3961 2945 ALCOHOL DEHYDROGENASE (EC 1.1. 1. 1) 217 218 RXA02539 CR00726 11676 10159 ALDEHYDE DEHYDROGENASE (EC 219 220 RXN03061 WV0034 108 437 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 221 222 RXN03150 W0155 10678 10055 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 223 224 RXN01340 W0033 3 860 ALOEHYDE DEHYDROGENASE (EC 1.2.1.3) 225 226 RXN01498 W0008 1598 3160 ALOEHYDE DEHYDROGENASE (EC 1.2.1.3) 227 228 RXN02674 WV0315 15614 14163 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) 229 230 RXN00868 W0127 2230 320 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) 231 232 RXN01143 W0077 9372 8254 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) 233 234 RXN01146 W0264 243 935 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) 235 236 RXNOI1144 WV0077 8237 7722 ACETOLACTATE SYNTHASE SMALL SUBUNIT (EC 4.1.3.18) Butanediol, diacetyl and acetoin formation Nucleic Acid Amino Acid Identification Code Contig.

SEQ ID NO SEQ ID NO 237 238 RXA02474 CR007 239 240 RXA02453 CR007 241 242 RXS01758 'xoii1 NT Start NT Stop Function 15 8082 7309 (S,S)-butane-2,3-dioi dehydrogenase (EC 1. 1. 1.76) 10 6103 5351 ACETOIN(DIACETYL) REDUCTASE (EC 1115 2 27383 28399 ALCOHOL DEHYDROGENASE (EC 1. 1.1. 1) 2007203039 29 Jun 2007 Table 1 (continued) H MP -Cycle Nucleic Acid SEQ ID NO 243 245 247 249 Amino Acid SEQ ID NO 244 246 248 250 Identification Code Contig. NT Start NT Stop Function RXA02 737 RXA02738 RXA02739 RXA00965 GR00763 3312 1771 GLUCOSE-6-PHOSPHATE 1-DEHYDROGENASE (C 1.1.1.49) GR00763 4499 3420 TRANSALIDOLASE (EC 2.2.1.2) GR00763 6769 4670 TRANSKETOLASE (EC 2.2.1.1) GR00270 1232 510 6-PHOSPH-OGLUCONATE DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44) W0106 2817 1366 6-PHOSPHOGLUCONATE DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44) GR00283 3012 4448 6-PHOSPHOGLUCONATE DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44) 251 252 RXN00999 253 254 F RXA00999 Nucleotide sugar conversion Nucleic Acid Amino Acid SEQ ID NO SEQ ID NO Identification Code RXN02596 F RXA02596 F R.A62642 RXA02572 RXA02485 RXA0 12 16 RXA0 1259 R.XA02028 RXA01262 RXA01 377 RXA02063 RXN00014 F RX(AOOO 14 RXAO1 570 RXA02666 RXA00825 Cont.

WV0098 GR00742 GR00749 GR00737 GROO7 18 GR00352 GR00367 GROO6 16 GR00367 GROO400 GR00626 WV0048 GROO002 GR00438 GR00753 GR00222 NT Start NT Stop Function 48784 5383 2 2345 2302 987 573 83511 3935 3301 8848 4448 427 7260 222 47582 489 5880 646 3445 1202 130 998 7191 5020 4527 9627 5227 1281 6493 1154 UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UDP.GALACTOPYRANOSE MUTASE (EC 5A4.99.9) UDP.GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UDP-GLUCOSE 6-DEHYOROGENASE (EC 1.1.1.22) UDP-N.ACETYLENOLPYRUVOYLGLUCOSAMINE REDUCTASE (EC 1.1.1.158) UDP-N.ACETYLGLUCOSAMINE PYROPHOSPHORYLASE (EC 2.7.7.23) UTP--GLUCOSE-1 -PHOSPHATE URIDYLYLTRANSFERASE (EC 2.7.7.9) UTP-GLUCOSE-1 -PHOSPHATE URIDYLYLTRANSFERASE (EC 2.7.7.9) GOP-MANNOSE 6-DEHYDROGENASE (EC 1.1.1.132) MANNOSE-1 -PHOSPHATE GUANYLTRANSFERASE (EC 2.7.7.13) GLUCOSE-I -PHOSPHATE ADENYLYLTRANSFERASE (EC 2.7.7.27) GLUCOSE-i -PHOSPHATE THYMIDYLYLTRANSFERASE (EC 2.7.7.24) GLUCOSE-i -PHOSPHATE THYMIDYLYLTRANSFERASE (EC 2.7.7.24) GLUCOSE-i -PHOSPHATE THYMIDYLYLTRANSFERASE (EC 2.7.7.24) D-RIBITOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC 2.7.7.40) DTDP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) Inositol and ribitol metabolism Nucleic Acid SEQ WD NO 287 Amino Acid SEQ ID NO 288 Identification Code Cat g NT Start NT Stop Function RXA01887 RA187 GR00539 4219 3209 MYD-INOSITOL 2-DEHYOROGENASE (EC 1.1.1.18) 2007203039 29 Jun 2007 Table 1 (continued) Nucleic Acid SEQ ID NO 289 291 293 295 297 299 301 303 305 307 309 311 313 Amino Acid SEQ ID NO 290 292 294 296 298 300 302 304 306 308 310 312 314 Identification Code RXN0001 3 F RKAOOOI3 RXA01 099 RXN01 332 F R-XIA01332 RXAO1 632 RKAOI 633 RXN01 406 RXN01 630 RXN00528 RXN03057 F RXA02902 RXA00251 Coniig VV0048 GROO002 GR00306 VV0273 GR00388 GR00454 GR00454 VV0278 WO0050 VV0079 W0028 GRI 0040 GRO0038 NT Start NT Stop 7966 3566 6328 579 552 2338 3380 2999 48113 23406 7017 10277 931 8838 4438 5504 4 4 33,42 4462 1977 47037 22318 7688 10948 224 Function MYO-INOSITOL-1 (OR 4)-MONOPHOSPH-ATASE 1 (EC 3.1.3.25) MYO-INOSITOL-1 (OR 4)-MONOPHOSPHATASE 1 (EC 3.1.3.25) INOSITOL MONOPHOSPHATE PHOSPHATAS 6 MYO-INOSITOL 2-DEHYDROGENASE (C 1. 1. 1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC1..18 MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC 1. 1. 1.18) MYO'-INOSITOL 2-DEHYDROGENASE (EC 1.18) MYO-iNOSITOL 2-DEHYDROGENASE (EC 1. 1. 1.18) MYO-INOSITOL-I -PHOSPHATE SYNTHASE (EC 5.5.1.4) myO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1-.18) GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) RIBITOL 2-DEHYDROGENASE (EC 1.1.1.6).

Utilization of sugars Nucleic Acid SEQ ID NO 315 317 319 321 323 325 327 329 331 Amino Acid SEQ ID NO 316 318 320 322 324 326 328 330 332 Identification Code RXN02654 F RXA02654 R.XNO1O49 F RXAO 1049 F RXA01050 RXA00202 RXN00872 F RXA00872 RXN00799 333 334 F RXA00799 RXA00032 RXA02528 RXN00316 F RXA00309 GR00752 W0079 GR00296 GR00296 GR00032 W0O127 GR00240 VV0009 GR00214 GROO003 GR00725 W000O6 GR00053 W0006 GROO053 GROO007 GROO61 5 GR00626 12206 13090 GLUCOSE 1-DEHYDROGENASE (E0 1.1.1.47) 7405 8289 GLUCOSE 1-DEHYDROGENASE 11 (EC 1.1.1.47) 9633 11114 GLUCONOKINASE (EC 2.7.1.12) 1502 492 GLUCONOKINASE (EC 2.7.1.12) 1972 1499 GLUCONOKINASE (EC 2.7.11A2) 1216 275 D-RII3OSE-BINDING PERIPLASMIC PROTEIN PRECURSOR 6557 5604 FRUCTOKINASE (EC 2.7.1.4) 565 1086 FRUCTOKINASE (EC 2.7.1.4) 5a.477 56834 PERIPLASMIC BETA-GLUCOSIDASEJBETA-XYLOSIDASE PRECURSOR (EC 3.2.1.21) (EC 3.2.1.37) 1 1584 PERIPLASMIC BETA-GLUCOSIDASEIBETA-XYLOSIOASE PRECURSOR (EC 3.2. 1.21) (EC 3.2.1.37) 12028 10520 MANNITOL 2-DEHYDROGENASE (EC 1. 1. 1.67) 6880 7854 FRUCTOSE REPRESSOR 7035 8180 Hypothetical Oxidoreductase (EC 1. 1. 316 5 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) 6616 7050 GLUCOSE--FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1. 99.2 8) 735 301 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) 1246 5 SUCROSE-6-PHOSPHATE HYOROLASE (EC 3.2.1.26) 725 6 SUCROSE-6-PHOSPHATE HYOROLASE (EC 3.2.1.26) 1842 349 SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) NT Start NT Stop Function 343 3.44 RXN00310 3-45 346 F RXAG0310 RXAOOO4I RX.A02 026 RXAO2061 2007203039 29 Jun 2007 Table 1 (continued) Nucleic Acid Amino Acid Identification Code Conrg NT Start NT Stop Function SEQ ID NO SEQ ID NO 353 354 RXN01369 WV0124 595 1776 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8) 355 356 F RXA0 1369 GR00398 3 503 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8) 357 358 F RXA01373 GR00399 595 1302 MANNOSE-6-PH-OSPHATE ISOMERASE (EC 5.3.1.8) 359 360 RY-A0261 1 GR00743 1 1752 1,4-A.LPI-A-GLUCAN BRANCHING ENZYME (EC 2.4.1.18) 361 362 RXA02612 GR00743 1793 3985 1.4-ALPHA-GLUCAN BRANCHING ENZYME (EC 2.4.1.18) 363 364 RXN01 884 WV0184 1 1890 GLYCOGEN DEBRANCHING ENZYME (EC 2.4.1.25) (EC 3.2.1.33) 365 366 F RXA01884 GR00539 3 1475 GLYCOGEN DEBRANCHING. ENZYME (EC 2.4.1.25) (EC 3.2.1.33) 367 368 RXA01111 GR00306 16981 17427 GLYCOGEN OPERON PROTEIN GLGX (EC 3.2. 369 370 RXN01550 WV0143 14749 16260 GLYCOGEN PHOSPHORYLASE (EC, 2.4.1.1) 371 372 F RXAOI1550 GR00431 3 13A6 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 373 374 RXN02IOO WV0318 2 2326 GLYCOGEN PHOSPHORYLASE (EC 2.4. 1. 1) 375 376 F RXA02100 GR00631 3 920 GLYC6GEN PHOspHORYLASE (EC 2.4.1.1) 377 378 F RXA02113 GR00633 2 1207 GLYtCOdtN PHOSPH0*R*YLASE (EC 2,4.1.1) 379 380 RXA02147 GR00639 15516 16532 ALPHA-AMYLASE (EC 3.2.1.1) 381 382 RXA01478 GROO422 10517 12352 GLUCOAMYLASE Gi AND G2 PRECURSOR (EC 3.2.1.3) 383 384 RXA01888 GR00539 4366 4923 GLUCOSE-RESISTANCE AMYLASE REGULATOR 385 386 RXN01927 W0O127 50623 49244 XYL.ULOSE KINASE (EC 2.7.1.17) 387 388 F RXA01927 GR00555 3 1118 XYLULOSE KINASE (EC 2.7.1.17) 389 390 RXA02729 GR00762 747 4 RIBOKiNASE (EC 2.7.1.15) 391 392 RXA02797 GR00778 1739 2641 RIBOKINASE (EC 2.7.1.15) 393 394 RXA02730 GR00762 1768 731 RIBOSE OPERON REPRESSOR 395 396 RXA02551 GR00729 2193 2552 6.PH OSPHOBETAGLUCOSIDASE (EC 1.2.1.86) 397 398 RX012 GRA,0385 5676 5005 utuYRIBO S-rtuSPHTr1 L OOLASE (EC 4.1.2.4) 399 400 RXAOO 195 GROO030 543 1103 1 -deoxy-O-xylulose 5-phosphate reduclolsomerase (EC 1. 1. 401 402 RXAOO196 GROO030 1094 1708 1 -deoxy-D-xylulose 5-p.hosphate reductoisomerase (EC 1. 1.1l.-) 403 404 RXN0I 562 W0191 1230 3137 1-DOGXYXYLULOSE-5-PHOSPHATE SYNTHASE 405 406 F RXA01562 GR00436 2 1039 1.DEOXYXYLULOSE-5 PHOSPHATE SYNTHASE 407 408 F RX(AOI1705 GR00480 971 1573 1-OEO6XYXYLULOSE-5.-PHOSPIIATE SYNTHASE 409 4,10 RXN00879 WV0099 8763 6646 4-ALPHA-GLUCANO TgANSFERASE (EC.2.4.1.25).

411 412 F RXA60879 GR00242 5927 3828 4-LH-LCNTA.FR~:(Ci4125), amylomaftase 413 414 RXN00043 WOI1S 3244 2081 N-ACETYLGLUCOS AMINE-6-P .HOSPHATE DEACETYLASE (EC 3.5.1.25) 415 416 F RXA00043 GROO007 3244 2081 N.ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE (EC 3.5.1.25) 417 418 RXN01752 WV0127 35265 33805 N.ACETYLGLUCOSAMINYLTRANSFERASE (EC 419 420 F RXAO1 839 GROO520 1157 510 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 421 422 RXA01859 GR00529 1473 547 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 423 424 RXA00042 GROO007 2037 1279 GLUCOSAMINE-6-PHOSPHATE ISOMERASE (EC 5.3.1.10) 425 426 RXA01482 GR00422 17271 15397 GLUCOSAMINE--FRUCTOSE-6-PHOSPHATE AM)NOTRANSFERASE (ISOMERIZING) (EC 2.6.1.15) 427 428 RXN03179 WV0336 2 667 URONATE ISOMERASE (EC 5.3.1.12) 429 430 F RXA02872 GR10013 675 4 URONATE ISOMERASE, Glucuronate Isomerase (EC 5.3.1.12) 431 432 RXN03180 WV0337 672 163 IJROtNATE ISOMERASE (EC 5.3.1.12) 433 434 F RXA02673 GR10014 672 163 URONATE ISOMERASE, Giucumonate Isomerase (EC 5.3.1.12) 435 436 RXA02292 GR00662 1611 2285 GALACTOSIDE O-ACETYLTRANSFERASE (EC 2.3.1.18) 437 438 RXA02666 GR00753 7260 64 '93 D-RiOITOL-5-PHOSPHATE CYTI DYLYLTRANSFE RASE (EC 2.7.7.40) 439 440 RXA00202 GR00032 1216 275 0-RI BOSE -BINDING PERIPLASMIC PROTEIN PRECURSOR 441 442 RYA02440 GR00709 5097 4258 0-RIBOSE-BINDING PERIPLASMVIC PROTEIN PRECURSOR 2007203039 29 Jun 2007 Table I (Continued) Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function SEQ ID NO SEQONO 443 4"4 RXN01569 W0009 41086 42444 dTDP-4-OEHYORORI-AMNOSE REDUCTASE (EC 1.1.1.133) 445 446 F RX(A01569 GR00438 2 427 DTDP-DEHYDRORHAMNOSE REDUCTASE (EC 1. 1. 1.133) 447 4-48 F RXA02055 GR00624 7122 8042 DTOP-4-DEH-YDRORHAMNOSE REDUCTASE (EC 1.1.1.133) 449 450 RXA00825 GR00222 222 1154 DTDP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) 451 452 RXA02054 GR00824 6103 7119 DTOP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) 453 454 R.XN00427 WVO 112 7004 6219 dTDP-RHAMNOSYL TRANSFERASE RFBF (EC 455 456 F RXA00.427 GROO098 1591 2022 DTDP-RH4AMNOSYL TRANSFERASE RFBF (EC 457 458 RXA00327 GR00057 10263 9880 PROTEIN ARAJ 459 460 RXA00328 GRoOOS7 11147 10656 PROTEIN ARAJ 461 462 RYA00329 GRO6057 12390 11167 PROTEIN'JARAJ 463 464 RXN01554 W01 35 28686 .26545 GLUCAN ENDO- 1.3-BETA-GLUCOSIOASE Al PRECURSOR (EC, 3.2.1.39) 465 466 RXN03015 WV0063 289 8 UDP-GLU CO.SE 6 -DEHY DROGENASE (EC 1.1.1.22) 467 468 RXN030_56 WV0028 6258 6935 PUTATIVE H EXULOSE-6-PHOSPHATE ISOMERASE (EC 46 470 RXN03030 WV0009 57006 56443 PERIPLASMIC B3ETA-GLUCOSIDASEIB3ETA-XYLOSIDASE PRECURSOR (EC 3.2.1.21) (EC 3.2.1.37) 471 472 RXN00401 W0025 12427 11489 5-DE~HYDRO-4-DEOXYGLUCARATE DEHYDRATASE (EC 4.2.1.4 1) 473 474 RXNO2 125 WV0102 23242 22442 AL60SE REDUCTASE (EC 1.1.1.21) 475 476 RXN00200 VVOI8I1 1679 5116 arabinosyl transferase subunit B (EC 477 478 RXN011175 W0017 39688 38303 PHOSPHO-2-DEHYDRO3.DEO(YHEPTONATE ALDOLASE (EC 4.1.2.15) 479 480 RXN01316 WV0091 561 10 4750 PUTATIVE GLYCOSYL TRANSFERASE WBIF 481 482 RXN01631 WO00S0 47021 46143 PUTATIVE HEXULOSE-6-PHOSPHATE ISOMERASE (EC 483 484 RXN01 593 WV0229 13274 12408 NAGO PROTEIN 45486 RXN00337 WV0197 20369 21418 GALACTOKINASE (EC 2.7.1.6) 487 488 RXS00584 W0323 5516 6640 PHOSPHO-2-D EHYDRO-3-DEOXYHEPTONATE ALDOLASE (EC 4.1.2.15) 489 490 RXS02574 BETA-HEXOSAMINIDASE A PRECURSOR (EC 3.2.1.52) 491 492 RXS032 15 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) 493 494 *F RXA01915 GROO549 1 1008 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) 495 496 RXS03224 CYCLOMAILTODEXTRINASE (EC 3.2.1.54) 497 498 F RX(A60O38 GROO006 1417 260 CYCLOjMALTOQE.XtRiNASE (EC 3.2.1.54) 499 500 RXC66233 protein involved in sugar metabolism 501 502 RXCOO236 Membrane Lipoprotein Involved in sugar metabolism 503 504 RXCO0271 Exported Protein involved in ribose metabolism 505 506 RXC00338 protein Involved in sugar metabolism 507 508 RXCO0362 Membrane Spanning Protein Involved In metabolism of diols 509 510 RXC00412 Amino Acid ABC Transporter ATP.Blnding Protein Involved in sugar metabolism 511 512 RXCO0526 ABC Transporter ATP-Binding Protein Involved In sugar metabolism 513 514 RXCOIOO4 Membrane Spanning Protein involved in sugar metabolism 515 516 RXCO1O17 Cytosolic Protein involved In sugar metabolism 517 518 RXCO1 021 Cytosolic Kinase Involved in metabolism of sugars and thiamin 519 520 RXCO1212 ABC Transporter ATP-Binding Protein involved In sugar metabolism 521 522 RXCO1306 Membrane Spanning Protein involved in sugar metabolism 523 524 RXCO1 366 Cytosolic Protein Involved in sugar metabolism 525 526 RXC01372 Cytosolic Protein Involved In sugar metabolism 2007203039 29 Jun 2007 Table i (continued) Nucleic Acid SEQ ID NO 527 529 531 533 535 537 539 541 Amino Acid SEQ ID NO 528 530 532 534 536 538 540 542 Identification Code g2n211& NT Start NT Sto- Function RXC01 659 RXC0 1663 RXCO 1693 RXCO 1703 RXC02254 RXC02255 RXC02435 F RXA02435 RXC03216 protein Involved in sugar metabolism protein Involved in sugar metabolism protein Involved in sugar metabolism Cytosolic Protein involved in sugar metabolism Membrane Associated Protein involved in sugar metabolism Cytosolic Protein involved in sugar metabolism protein Involved in sugar metabolism 268 Unchiaracterized protein Involved in glycerol metabolism (homolog of Drosophila rhomboid) protein involved in sugar metabolism GR00709 825 543 544 TCA-cycle Nucleic Acid SEQ ID NO 545 547 549 551 553 555 557 559 561 563 Amino Acid SEQ ID NO 546 548 550 552 554 556 558 560 562 564 Identification Code RXA021 75 RXA02621 RXN00519 F 0"00O521 RXN02209 F RXA02209 RXN02213 F RXA02213 RXA02056 RXA0 1745 RXA00782 RXA00783 RXN0 1695 F RXA01615 F RXA01695 RXA00290 RXN0 1048 F RXA01O48 F RXA00290 RX NO31 01 conl GROO641 GR00746 W01 44 GROO1 33 W0304 GR00648 WV0305 GR00649 GR00625 GR00495 GR00206 GR00206 W01I39 GR00449 GR00474 GR00046 W0079 GR00296 GR00046 WV0066 W0025 W0025 107 10 2647 5585 2 1378 1330 3 2 3984 5280 11307 8608 4388 4693 12539 3 4693 2 15056 11481 9418 1829 3372 1060 1671 1661 2151 2046 2870 1495 3103 4009 12806 9546 4179 5655 11316 290 5655 583 14640 9922 NT Start -NT Stop Function CITRATE SYNTHASE (EC 4.1.3.7) CITRATE LYASE BETA CHAIN (EC 4.1.3.6) ISOCITRATE DEHYDROGENASE (NADP) (EC 1.1.1.42) ISOCITRATE-D)EHYDROGENASE [NADPI (EC 1.1.1.42) ACONITATE HYDRATASE (EC 4.2.1.3) ACONITATE HYIDRATASE (EC 4.2.1.3) ACONITATE HYDRATASE (EC 4.2.1.3) ACONITATE HYDRATASE (EC 4.2.1.3) 2-OXOGLUTARATE DEHYDROGENASE El COMPONENT (EC 1.2.4.2) DIHYDROLIPOAMIOE SUCCINYLTRANSFERASE COMPONENT (E2) OF 2-OXOGLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.6 1) SUCCINYL-OA SYNTI-ETASE ALPHA CHAIN (EC 6.2.1.5) SUCCINYL-COA SYNTHETASE BETA CHAIN (EC 6.2.1.5) L-MAI.ATE IDEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) L-MALATE DEHY(DROGENASE (ACCEPTOR) (EC 1.1.99.16) L-MALATE DEHYIDROGENASE (ACCEPTOR) (EC 1.1.99.16) MALIC ENZYME&(EC 1. 1. 1.39) MALIC ENZYME (EC 1.1.1.39) MALIC ENZYMVIE (EC-1.1.1.39) MALIC ENZYME (EC 1.1.1.39) DIHYDROLIPOAMIDE SUCCINYLTRANSFERASE COMPONENT (E2) OF 2-OXOGLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.61) IJIHYDROLIPOAMIDE SUCCINYLTRANSFERASE COMPONENT OF 2- OXOGLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.61) oxogluta rate semialdehyde dehydrogenase (EC 1.2. 585 586 RXN02046 587 588 RXN00389 2007203039 29 Jun 2007 Glyoxylate bypass Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 589 591 593 595 597 599 Amino Acid Identification Code SEQ ID NO 590 RXN02399 592 IF RXA02399 594 RXN02404 596 F RXA02404 598 RXA01089 600 RXA0186 C Onlg W0176 GR0D699 W0O176 GR00700 GR00304 GROO539 19708 478 20259 3798 3209 3203 18365 1773 22475 1663 3955 2430 ISOCITRATE LYASE (EC 4.1.3.1) ISOCITRATE LYASE (EC 4.1.3.1) MALATE SYNTHASE (EC 4.1.3.2) MALATE SYNTHASE (EC 4.1.3.2) GLYOXYLATE-INDUCED PROTEIN GLYOXYLATE-INDUCED PROTEIN Methylcitrate-pathway Nucleic Acid Amino Acid Identification Code SEQ ID NO SEQ ID NO 600 602 RXN03117 601 604 F RXA00406 603 606 F RXA00514 605 608 RXA00512 607 6 10 RXA00518 609 612 RXA01077 611 614 RXN03144 613 616 IFRXA02322 615 618 RXA02329 617 620 RXA02332 619 622 RXN02333 621 624 F RXA02333 623 626 RXA00030 Methyl-Malonyl-CoA-Mutases Contig W0092 GROO090 GROO130 GROO 130 GROQ131 GRO0300 W01 41 GR00668 GR00669 GR00671 WV0141 GR00671 GROO003 NT Start NT Stop Function 1576 4 1576 4 2773 6017 901 5 5 764 1815 1902 9979 2-methylisocitrate synthase (EC 2-methytisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylcltrate synthase (EC 4.1.3.31) 2-methylcitrate synlhase (EC 4.1.3.31) 2-methylisocitrate synthase (EC 2-mothylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylisocitiate synthase (EC 2-methylcitrate synlhase (EC 4.1.3.31) methylisocitrate lyase (EC 4.1.3.30) methylisocitrate lyaso (EC 4.1.3.30) LACTOYLGLUTATIONE LYASE (EC 4.4.1.5) Nucleic Acid SEQ ID NO 625 627 629 Amino Acid Identification Code SEQ ID NO 628 RXN00148 630 F RXAOO 148 632 RXA00149 Coig.

WV0167 GR00023 GROO023 NT Start NT Stop Function 12059 5 2009 METHYLMALONYL-COA MUTASE ALPHA-SUBUNIT (EC 5.4.99.2) METHYLMALONYL-COA MUTASE ALPHA-SUBUNIT (EC 5.4.99.2) METHYLMALONYL-COA MUTASE BETA-SUBUNIT (EC 5.4.99.2) 2007203039 29 Jun 2007 Table I (continued) Others Nucleic Acid Amino Acid Identification Code oni. NT Start NT Slop Function SEQ ID NO SEQ ID NO 631 634 RXN00317 W0197 26879 27532 PHOSPHOGLYCOLATE PHOSPH-ATASE (EC 3.1.3.18) 635 636 F RXA00317 GR00055 344 6 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) 637 638 PXA02196 GR00645 3956 3264 PI-OSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3. 18) 639 640 RXN02461 W0124 14236 14643 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) Redox Chain Nucleic Acid Amino Acid Identification Code Cni. NT Start NT Stop Function SEQ ID NO SEQ ID NO 641 642 RXN01 744 W0O174 2350 812 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 15A3 644 F RXA00055 GRO0008 11753 11890 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 1545 646 F RXA01744 GR00494 2113 812 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 647 648 RXA00379 GRO0082 212 6 CYTOCHROME C-TYPE BIOGENESIS PROTEIN CODA 649 650 RXA00385 GR00083 773 435 CYTOCHROME C-TYPE BIOGENESIS PROTEIN CODA 651 652 RXA01 743 GR00494 806 6 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT 11 (EC 1.10.3.-) 653 654 RXN02480 WV0084 31222 29567 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 655 656 F RXA01919 GROS550 288 4 CYTOCHROME C OXIDASE SUBUNIT I (EC 1.9.3.1) 657 658 F RXA02480 GR00717 1449 601 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 659 660 F RXA02481 GR00717 1945 1334 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 661 662 RXAO2140 GR00639 7339 8415 CYTOCHROME C OXIDASE POLYPEPTiDE 11 (EC 1.9.3.1) 663 664 RXA02142 GR00639 9413 10063 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 655 666 RXA021.44 GR00639 11025 12248 RIESKE IRON-SULFUR PROTEIN 667 668 RXA02740 GR00763 7613 8542 PROBABLE CYTOCHROME C OXIDASE ASSEMBLY FACTOR 669 670 RXA02743 GR00763 13534 12497 CYTOCHROME AA3 CONTROLLING PROTEIN 671 672 RXA01227 GR00355 1199 1519 FERREDOXIN 673 674 RXA01865 GR00532 436 122 FERREDOXIN 675 676 RXA00680 GR00179 2632 2315 FERREDOXIN VI 677 678 RXA00679 GR00179 2302 1037 FERREDOXIN--NAO(+) REDUCTASE (EC 1.18.1.3) 679 680 RYA00224 GR00032 24965 24015 ELECTRON TRANSFER FLAVOPROTEIN ALPHA-SUBUNIT 631 682 RXA00225 GR00032 25783 24998 ELECTRON TRANSFER FLAVOPROTEIN BETA-SUBUNIT 683 684 RXN00606 W0192 11299 9026 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 685 686 F RXA00606 GROO160 121 1869 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 687 688 RXN00595 W0192 8642 7113 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 689 690 F RXA00608 GROO160 2253 3017 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 691 692 RXAOO913 GR00249 3 2120 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 693 694 RXA00909 GR00247 2552 3406 NADH DEHYDROGENASE I CHAIN L (20 1.6.5.3) 695 696 RYA00700 GRO0182 846 43 NADH-UBIQUINONE OXIDOREDUCTASE CHAIN 2 697 698 RXN00483 W0086 44824 46287 NADH-UBIQUINONE OXIDOREDUCTASE 39 KD SUBUNIT PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) 2007203039 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 699 Amino Acid SEQ ID NO 700 Identification Code F RXA00483 RXA0 1534 RXA00288 RXA02741 RXN02560 F RXA02560 RXA0 1311 RXN03014 F RXA0091I0 RX N01895 F RXA01895 RXA00703 RXN00705 F RXAOO7O5 RXN00388 F RXA00388 F RXA00386 Conati GRO01 19 GR0D427 GROO046 GR00763 w0l01 GR00731 GROO380 W0058 GR00248 WVO 117 GR00543 GROD1 83 W0005 GRO0184 W0025 GROO085 GR00084 GR00259 W0101 GR00731 GR00408 GR00214 GR00639 W0058 W0176 W03 17 W0302 wolol1 19106 1035 2646 9585 9922 6339 1611 1273 3 955 2 2556 6111 1291 2081 969 514 1876 5602 2019 2297 2031 10138 405 32683 3552 1784 4633 20569 547 1636 8620 10788 7160 865 368 1259 5 817 271 5197 407 3091 667 5 2847 6759 3176 3373 3134 11025 4 33063 2794 8.49 4010 NAOI--UBIQUINONE OXIDOREDUCTASE 39 KID SUBUNIT PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) NADH-DEPENOENT FMN OXYDOREDUCTASE QUINONE OXIDOREDUCTASE (EC 1.6.5.5) QUINONE OXIDOREDUCTASE (EC 1.6.5.5) NADPH-FL.AVIN OXIDOREDUCTASE (EC 1.6.99.-) NAOPH-FLAVIN OXIDOREDUCTASE (EC 1.6.99.-) SUCCINATE DEHYDROGENASE IRON-SULFUR PROTEIN (EC 1.3.99.1) NADH DEH-YDROGENASE I CHAIN M (EC 1.6.5.3) Hydrogenase subunits NADH DEHYOROGENASE (EC 1.6.99.3)

DEI-YROGENASE

FORMATE DEHYDROGENASE ALPHA CHAIN (EC 1.2.1.2) FDHD PROTEIN FDHD PROTEIN CYTOCHROME C BIOGENESIS PROTEIN CCSA essential protein similar to cytochrome c RESC PROTEIN, essential protein similar to cytochrome c biogenesis protein putative cytochrome oxldase FLAVOHEMOPROTEIN DIHYDROPTERIDINE REDUCTASE (EC 1.6.99.7)

FLAVOHEMOPROTEIN

GLUTATHIONE S-TRANSFEPASE (EC 2.5.1.18) GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE (EC 1.2.1.1) OCRC PROTEIN, men aquinol:cytochrome c oxidoreductase NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) NADH-UBIOUINONE OXIDOREDUCTASE CHAIN 4 (EC 1.6.5.3) Hypothetical Oxidorductase Hypothetical Oxidoreductase H-ypothetical Oxidoreductase (EC 111.

733 734 RXA00945 735 736 RXN02556 F RXA02556 RXA01 392 RXA00800 RXA02143 RXN03096 RXN02036 RXN02765 RXN02206 RXN02554 ATP-Synthase Nucleic Acid SEQ ID NO 755 757 759 761 763 765 Amino Acid SEQ ID NO 756 758 760 762 764 766 Identification Code RXN01 204 F RXAO12O4 RXA01201 RXNO 1 '193 F RXAOI 193 F RXAO 1203 o.

W0 121 GR003,45 GRO0344 W0175 GRO0343 GR00344 NT Start NT Slop Function 1270 394 675 5280 15 3355 ATP SYNTHASE A CHAIN (EC 3.6.1.34) ATP SYNTHASE A CHAIN (EC 3.6.1.34) ATP SYNTHASE ALPHA CHAIN (EC 3.6.1.34) ATP SYNTHASE BETA CHAIN (EC 3.6.1.34) ATP SYNTHASE BETA CHAIN (EC 3.6.1.34) ATP SYNTHASE BETA CHAIN (EC 3.6.1.34) 2007203039 29 Jun 2007 Table 1 (continued) NT Start NT-on Fuc~o Nucleic Acid SEQ ID NO 767 769 771 773 775 777 Amino Acid Identification Code Confie.

SEQ ID NO 768 RXN02621 WV0121 770 IF RXA02821 GROW~ 772 RXA01200 GROO3 774 RXA01 194 GRO03 776 RXA01202 GRO03 778 RXN02434 %A/nnoe 02 4-4 43 44 85 318 610 1141 3349 3274 ATP SYNTHASE C CHAIN (EC 3.6.1.34) ATP SYNTHASE C CHAIN (EC 3.6.1.34) ATP SYNTHASE DELTA CHAIN (EC 3.6.1.34) ATP SYNTHASE EPSILON CHAIN (EC 3.6.1.34) ATP SYNTHASE GAMMA CHAIN (EC 3.6.1.34) ATP-8INDING PROTEIN Cytochrome metabolism Nucleic Acid SEQ ID NO 779 781 Amino Acid Identification Code Cnt.

SEQ ID NO 780 RXN00684 WV0005 782 RXN'00387 WV0025 NT Start NT Stop Function 29864 28581 1150 2004 CYTOCHROME P450 116 (EC Hypothetical Cytochromne c Biogenesis Protein 2007203039 29 Jun 2007 2 -Excluded Genes GenBank Tk Gene Name Gene Function Reference Accession No.

A09073 ppg Phosphoenol pyruvate carboxylase Bachrnann, B3. 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, B3. 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 AB003 132 murC; ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and characterization of the ftsZ gene from coryneform bacteria," Biocliem. Biophys. Res. Comm un., (1997) ABO 15023 murC; flsQ Wachi, M. et al. "A murC gene from Coryneform bacteria," App!. Microbiol.

13o*echnoL., 5)l(2):223-228 (1999) AB0 18530 dtsR Kimura, E. et al. "Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium lactofermen urn,' Biosci. Bicn'echno!. Biochem., 60(10): 1565-1570 (1996) AB018531 dtsR 1; dtsR2 AB020624 murl 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 gInA Glutamine synthetase AF030405 hisF cyclase AF030520 argG Argininosuccinate synthetase AF03 1518 argF Ornithine carbamolytransferase AF036932 aroD 3-dehydroquinate dehydratase AF038548 pyc Pyruvate carboxylase 2007203039 29 Jun 2007 __ap t,_r T a b le 2 (c o n t in u e d F 8655 ciE;ap;re 1 Dip-eptide-binding protein; adenine Wehmeier, L. et al. "The role nf the Corynebacterium g!utarnicum rcl gcn-c in phosphoribosyltransferase; GTP (p)ppGpp metabolism," Microbiology, 144:1853-1862 (1998) pyrophosphokinase A F0414 3 6 a rg-R Arginine repressor 7F-045998 -impA Inositol monophasphate phosphatase AF048764 argH Argininosuccinate lyase AF049897 argC; argi; argB; N-acetylglutamyl1phosphate -reductase;, argD; argF; argR; omnithine acetyhtransferase;

N-

argG; argH acetyiglutamate kinase; acetylornithine transminase; ornithine carbamoyl[trans ferase; arginime repressor; argininosuccinale synthase; argininosuccinate lyase AF050109 inhA Enoyl-acyl carrier protein reductase AF050 166 hisG ATP phosphoribosyicransferase AF05184 hisA P-hosphori bosyl form im ino5amino- I phosphoribosyl-4- im idazolecarboxam ide isomerase AFU52652 metA Homoserine 0-acetyl trans ferase Park. S. et al ktin and ~A AF053071I AF060558 A- FO86704 arc B hisH li1s Dehydroquinate synthetase Glutamine amidotransferase Phosphoribosyl-ATPpyrophosphohydrolase 5-enolpyruvylshikimate 3-phosphate synthase L-aspartate-alpha-decarboxyase precursor me ,L~LX a mtmniiiiiie Diosyntnetic gene encoding homoscrine acetyltransferase in Corynebacterium. glutamicum," Mo.

Cells., 8(3):286-294 (1998) AF1 14233 aroA AF1 16T8-4 panD Dusch, N. et al. "Expression of the Corynebaccerium giutamicumn panD gene encoding L-aspartate-al]ph a-decarboxy lase leads to pantothenate overproduction in Escherichia coli," App!. Environ. Microbiol. 65(4)1530- 1539 (1999)1 I A1 245 t8 aroD; aroE 3-dehydroquinase; shikimate dehydrogenase AF124600U aroC; aroK; aroB; pepQ Chorismate synthase; shikimate kinase; 3cytoplasmic nentidlase AF14587 nA AH456qja6 iflfA

I-

J

I

2007203039 29 Jun 2007 _Table 2 (continued) A.1001436 ectP 1Transport of ectoine, glycine betaine, 1Peter, 14. et "Corynebacterium glutamicumn is equipped with four secondary1 I I pralne carrir orempatibte col-les: Identification, sae-uenn, and characterizatonn 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 t role in cell wall integrity: A study with Corynebacterium glutamicum," J.

Bacterial., 180(12):3159-3165 (1998) AJO07732 ppc; secG; amt; ocd; Phosphoenolpyruvate-carboxylase; high soxA affinity ammonium uptake protein; putative omith ine-cyclodecarboxylase; sarcosine oxidase AJO 103 19 ftsY, glnB, glnD; srp; Involved in cell division; PH1 protein; Jakoby, M. et al. "Nitrogen regulatio'n in Corynebacteriumn glutamicum; amntP uridylyltransferase (uridylyl-removing Isolation of genes involved in biochemical characterization of corresponding enzmye); signal recognition particle; low proteins," FEMS Microbial, 1 73(2):303-3 10 (1999) affinity ammonium uptake protein AJ 132968 cat Chloramphen icol aceteyl transferase AJ224946 mqo L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical and genetic characterization of the mem brane-associated malate dehydrogenase (acceptor) from Corynebacterium glutamicum," Eur. J. Biochem., 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 formed by a low molecular mass polypeptide," Biochemistry, 37(43): 15024-15032 (1998) D17429 Transposable element IS31831 Vet-tes et al."lsolation and characterization of 1S3 183 1, a transposable element from Corynebacterium glutamicum," Mo. Microbial., 11l(4):739-746 (1994) D84 102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al. "Molecular cloning of the Corynebacterium glutamicumn (Brevibacterium lactofermentumn AJ 12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase," Microbiology, 142:3347-3354 (1996) EQ01358 hdh; hA Homoserine dehydrogenase; homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: JP 1987232392-A I 10/12/87 E01359 Upstream of the start codon of homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: JP kinase gene 1987232392-A 2 10/12/87 EQ01375 Tryptophan EQ 1376 trpL; trpE Leader peptide; anthranilate synthase Matsui, K. et al. "'Tryptophan operon, peptide and protein coded thereby, utilization of tryptophan operon gene expression and production of Patent: JP 1987244382-A 1 10/24/87 2007203039 29 Jun 2007 2 (continued) EQ' 1377 TPromotor andl opcrator regions off Ma"ui, K. ct opan operon, peptide and protcin ccdhcrcby, tryptophan operon utilization of tryptophan operon gene expression and production of tryptophan," Patent: JIP 1987244382-A 1 10/24/87 E03937 Biotin-synthase Hatakeyama, K. et al. "DNA fragment containing gene capable of coding synthetase and its utilization," Patent: JP 1992278088-A 1 10/02/92 E04040 Diamino pelargonic acid aminotransferase Kohama, K. et al. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1 E04041 Desthiobictinsynthetase Kohama, K. et at. "Gene coding diaminopelargonic acid aminotransferase and dlesthiobiotin synthetase and its utilization," Patent: JP 1992330284-A I 11/.18/92 E04307 Flavum aspartase Kurusu, Y. et at. "Gene DNA coding aspartase and utilization thereof," Patent: JP 1993030977-A 1 02/09/93 E04376 Isocitric acid lyase Katsumata, R. et at. "Genie manifestation controlling DNA," Patent: JP 1993056782- A 3 03/09/93 E04377 Isocitric adid lyase N-terminal fragment Katsumata, R. et al. "Gene mnanifestaition cotitrolling-DNA," Patent: JP 1993056782-A 3 03/09/93 E04484 Prephenate dehydratase Sotouchi, N. et at. "Production of L-phenylalanine by fermentation," Patent: JP 2 03/3093, EQS5108 Aspartokinase Fugono, N. et al. "Gene DNA coding Aspartokinase and its use," Patent: JP 184366-A 1 07/27/93 112 Dihiydro-dipichorinate synthetase Hatakeyama, K. et at. "Gene DNA coding dihydrodipicolinic acid synthetase and its use," Patent: JP 1,993 18437 1 -A 1 07/27/93 E05776 Diaminopimelic acid dehydrogenase Kobayashi, M. et at. "Gene DNA coding Diaminopimelic acid dehydrogenase and its use," Patent:.JR .19932984970-A '1 11/0&93 E05779 Threonine synthase Koharna, K. et al. "Gene-DNA coding threonine synthase and its use," Patent: JP 1993284972-A 1 1-1/02/93 E061 10 Prephenate dlehydratase Kikuchi, T. et at. "Production of L-phenylalanine by fermnentation method," Patent: JP 1993344881-A 1 12/27/93 E061 ll Mutated Prephienate dehydratase Kikuchi, T. et at. "Production of L-phenylatanine by fermentation method," Patent: JP 1993344881 I-A 1 12/27/93 E06 146- Acetohydroxy acid synthetase lnui, M. et at. "Gene capable of coding Acetohydroxy acid synthetase and its use," Patent: JP 1993344893-A 1 12/27/93 E06825 Aspartokinase Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A 1 03/08/94 E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A I 03/08/94 2007203039 29 Jun 2007 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 protein to membrane," Patent: JP 1994169780-A 1 06/21/94 E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08178, Feedback inhibition-released 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 E08180, E08181, E08182 E08232 Acetohydroxy-acid isomeroreductase Inui, M. et al. "Gene DNA coding acetohydroxy acid isomeroreductase," Patent: JP 1994277067-A .1 10/04/94 E08234 secE Asai, Y. et al. "Gene DNA coding fir translocation machinery of protein," Patent: JP 1994277073-A I 10/04/94 E08643 FT aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA fragment having promoter function in synthetase promoter region coryneform bacterium," Patent: JP 1995031476-A 1 02/03/95 E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having promoter function in coryneform bacterium," Patent: JP 1995031476-A 1 02/03/95 E08649 Aspartase Kohama, K. et al "DNA fragment having promoter function in coryneform bacterium," 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 I 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 E12594 Serine hydroxymethyltransferase Hatakeyama, K. et al. "Production of L-trypophan," Patent: JP 1997028391-A 1 02/04/97 E12760, transposase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: E12759, JP 1997070291-A 03/18/97 E12758 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 E12773 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 2007203039 29 Jun 2007 _Table2 cntinued)1 U136-35 Tilucose-6-phosphate dehlydrogenase CHatakcyama, K. ct "Glucosc-6-phosphatc dehydrogenasc an~d DNA capable I I of coding the same," Patent: JP 1997224661-A 1 09/02/97 LO 1508 INvA Thrconine dehydratase Moeckel, B. et al. "Functional and structural analysis of the threonine dehydratase of Corynebacterium glutamicum," J. !3acleriol., 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 glutam icumn 3-deoxy-D-arabinoh eptulosonate-7-phosphate synthase gene," Microbial LeIlf, 107:223-230 (1993) L09232 llvB; ilvN; ilvC Acetohydroxy acid synthase large subunit; Keilhauer, C. et al. "Isoleucine synthesis in Corynebacteriumn glutamicum: Acetohydroxy acid synthase small subunit; molecular analysis of the i~vB-ilvN-ilvC operon,".!. Bacterial., 175(17):5595- Acetohydroxy acid isomeroreductase 5603 (1993) L18874 PtsM Phosphoenolpyruvate sugar Fouet, A-et al. "Bacillus subtilis sucrose-specific enzyme I1 of the phosphotransferase phosphotransrerase 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 Left., 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. Biatechnal., (1994) L27 126 Pyruvate kinase Jetten, M. S. et al. "Structural and functional analysis of pyruvate kinase from Corynebacteriumn glutamicum," Appl. Environ. Milcrobial, 60(7):250 1-2507 L28760 aceA Isocitrate lyase L35906 dtxr Diphtheria toxin repressor Oguiza, J.A. et al. "Molecular cloning, DNA sequence analysis, and characterization of the Corynebacteriumn diphtheriae dtxR from Brevibacteriumn lactofermentum," J Bacterial., 1 77(2):465-467 (1995) M 13774 Prephenate dehydratase Follettie, M.T. et al. "Molecular cloning and nucleotide sequence of the Corynebacterium gluiamicum pheA BacteriaL, 167:695-702 (1986) M16175 5S rRNA Park, Y-H. et al. "Phylogenetic analysis of the coryneform bacteria by 56 rRNA sequences,".!. 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 Iactofcrrnentum, a glutamic-acid-producing bacterium," Gene, 52:191-200 (1987) *M16664 trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and function of the trp operon control regions of Brevibacterium lactofermentum, a glu tam ic-acid-produc ing bacterium," Gene, 52:191-200 (1987) 2007203039 29 Jun 2007 Table 2 (continued) M25819 Phosphoenolpyruvate carboxylase O'Regan, M. et al. "Cloning and nucleotide sequence of the Phosphoeno!pyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCCI3032," Gene, 77(2):237-251 (1989) M85106 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes," J Gen.

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

Microbiol., 138:1167-1175 (1992) M89931 aecD; brnQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, I. et al. "The Corynebacterium glutamicum aecD gene encodes a C-S uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination activity that degrades aminoethylcysteine," J. Bacteriol., 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product," Arch. Microbiol., 169(4):303-312 (1998) S59299 trp Leader gene (promoter) Herry, D.M. et al. "Cloning of the trp gene cluster from a tryptophanhyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence," Appl. Environ. Microbiol., 59(3):791-799 (1993) U 11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican, L.K. (1994) Complete nucleotide sequence of the Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology Department, University College Galway, Ireland.

U13922 cgllM; cgllR; cigliR Putative type 11 5-cytosoine Schafer, A. et al. "Cloning and characterization ofa DNA region encoding a methyltransferase; 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 Escherichia type III restriction endonuclease coli," J. Bacteriol, 176(23):7309-7319 (1994); Schafer, A. et al. "The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine in an McrBCdeficient Escherichia coli strain," Gene, 203(2):95-101 (1997) U14965 recA U31224 ppx Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., 178(15):4412-4419 (1996) U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., 178(15):4412-4419 (1996) U31230 obg; proB; unkdh ?;gamma glutamyl kinase;similar to D- Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., dehydrogenases 178(15):4412-4419 (1996) 2007203039 29 Jun 2007 2 (continued) U31281 TbinoB n iotin synthaSe S Sreb ii "Twyo new members of the bi- B superfamiiy: Cloninga, sequencing and expression of bio B genes of Methylobacillus flagellatumn and glutamicum," Gene, 175: 15-22 (1996) U35023 IhtR; accBC Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A Corynebacterium glutamicumn gene encoding a two-domain carboxylase protein similar to biotin carboxylases and biotin-carboxyl-car-rier proteins," Microbiol, 166(2);76-82 (1996)' U43535 cmr Multidrug resistance protein Jager, W. et al. "A Corynebacterium glutamicum gene conferring muitidrug resistance in the heterologous host Escherichia coli," I Bacteriol.

179(7):2449-2451I (1997) U43536 clpB Heat shock ATP-binding protein U53587 aphA-3 3'5"-aminoglycoside phosphotransferase U89648 Corynebacterium glutamicum unidentified sequence involved in histidine biosynthesis, partial sequence X04960 trpA; trpB; I-pC; IrpD; Tryptophan operon Matsui, K. et al. "Complete nucleotide and deduced amino acid sequences of trpE; trpG; trpL the Brevibacterium lactofermentumn tryptophan operon," Nucleic Acids Res., 14(24):101l13-101 14 (1986) X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al. "Nucleic sequence of the lysA gene of Corynebacterium decarboxylase, EC 4.1.1.20) glutamicum and possible mechanisms for modulation of its expression," Mo!.

Gen. Genet., 212(l):112-119 (1988) X14234 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," Mo& Gen. Genet., 218(2):330-339 (1989); Lepiniec, L. et al.

"Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function and molecular evolution," Plant. Mo!. Riot., 21 (3):487-502 (1993) X173 13 fda Fructose-bisphosphate aldotase 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," Mot. MicrobioL, X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al. "Nucleic sequence ofie dapA gene from Corynebacterium glutamicum," NucleicAcids Res., 18(21):6421 (1990) X54223 AttB-related site Cianciotto, N. et at. "DNA sequence homology between att B3-related sites of Corynebacteriumn diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicumn and the atIP site of lambdacorynephage," FEMS. Microbiol, Lei., 66:299-302 (1990) X54740 argS, lysA Arginyl-tRNA synthectase; Diaminopimetate Marcel, T. et al. "Nucleotide sequence and organization of the upstream region decarboxylase of the Corynebacterium glutamicum lysA gene," McI. MicrobioL, 4(11): 1819- 1830 (1990) 2007203039 29 Jun 2007 Table 2 (continued) X55994 trpL; trpE fPutative leader peptide; anthranilate Hecery, D.M. et al "Nucleotide sequence of the Corynebacterium glutamicumn Isynithase coloponlent I rpE gene," AMiclec- Acids Res., 18(23):713 8 (1990) X56037 thrC Threonine synthase Han, K.S. et al "The molecular structure of the Corynebacterium glutamicum threonine synthase gene," Mo!. Microbiol, 4(tO0): 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. Microbiol, 66:299-302 (1990) X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit; Kalinowski, J. et al. "Genetic and biochemical analysis of the Aspartokinase asd Aspartokinase-beta subunit; aspartate beta from Corynebacterium glutamicum," Mo!. Microbiol, 5(5):1 197-1204 (1991); semialdehyde dehydrogenase Kalinowski, J. el al. "Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspertate beta-semialdehyde dlehydrogenase gene asd in glutamicum," Mo!. Gen. Genet., 224.(3):3 17-324 (1990) X59403 gap;pgk; tpi 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 enzym es .glyceraidehyde-3 -phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomeras," J. Bacteriol, 174(1 9):6076-6086 (1992) X59404 gdh Glutamate dlehydrogenase Bormann, E.R. et al. "Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase," Mo!. Microbiol, 6(3):317-326 X60312 lysi L-lysine permease Seep-Feldhaus, A.H. et al. "Molecular analysis of the Corynebacterium glutamicum lysI gene involved in lysine uptake," Mo!. MicrobioL, 5(12):2995- 3005 (1991) X66078 cop I Psi protein Joliff, G. et al. "Cloning and nucleotidle sequence of the csplI gene encoding PSI1, one of the two major secreted proteins of Corynebacteriurn glutamicum: The deduced N-termninal region of PSI is similar to the Mycobacterium antigen complex," Mo! Microbiol, 6(16):2349-2362 (1992) X66 112 glt Citrate synthase Eikmnanns, BiJ. et al. "Cloning sequence, expression and transcriptional analysis of the Corynebacterium glutamicumn gItA gene encoding citrate synthase," Microbiol, 140:1817-1828 (1994) X697737 cspB Surface layer prote reutS2 Peyrct, J.L. et al. "Characterization of the cspB gene encoding PS2, an ordered X6913 cp2 urfce lyerproeinPS2surface-layer protein in Corynebacterium glutam icum," Mo!. Microbiol, 9(1):97-109 (1993) Dunrtl, CL l. U~l~u..uu~ u vv,0 X69 104 153 related insertion element 153-related insertion sequence and phylogenetic analysis," Mo!. Microbiol., 14(3):571-581 (1994) I 2007203039 29 Jun 2007 X7095 Table 2 (continued) X7099 IeA T Suprupyliiialat syiLandsu ratek, M. ci al. "Leucine synthesis in Corynebacterium glutamicurn: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis," Appi. Environ. Microbial, 60(l): 133-140 (1994) X71489 icd Isocitrate dlehydrogenase (NADP+) Eikmanns, B.J. et at. "Cloning sequence analysis, expression, and inactivation of the Corynebacteriumn glutamicumn icd gene encoding isocitrate dehydrogenase and biochemnical characterization of the enzyme," J. Bacleriol, (1995) X72855 GDHA Glutamate dehydrogenase (NA DP+) Xj75083, mtrA 5-m ethyl tryptophan resistance Heery, D.M. et al. "A sequence from a tryptophan-hyperproducing strain of X70584 Corynebacteriumn glutamicumn encoding resistance to Biochen. Biophys. Res. Cotumun., 201(3): 1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA mutant strains ofCorynebacterium glutamicum and Brevibacterium lactofermentum," App!.

Bioa'ecinol., 42(4):575-580 (1994) X75504 aceA; thiX Partial Isocitrate lyase; Reinscheid, D.J. et al. "Characterization of the isocitrate lyase gene from Corynebacterium glutamicumn and biochemical analysis of the enzyme," J.

176(12):3474-3483 (1994) X76875 ATPase beta-subunit Ludwig, W. et al. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factorTu 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 Antonie Van Leeuivenhoek 64:285-305 (1993) X77384 recA Billman-Jacobe, H. "Nucleotide sequence of a recA gene from ____________Corynebacteriumn glutamicum," DNA Seq.. 4(6):403-404 (1994) X78491 aceB Malate synthase Reinscheid, DiJ. et al. "Malate synthase from Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase: sequence analysis," Microbiology, 140:3099-3 108 (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, 14 1:523-528 (1995) X81 191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, WV. et al. "Structure of the gluABCD cluster encoding the glu D glutamate uptake system ofCorynebacteriumn glutamicum," J. Bacterial, 177(5):1 152-1158 (1995) X81379 dapE Succinyldliaminopimelate dlesuccinylase Wehrmann, A. et al. "Analysis of different DNA fragments of Corynebacteriumn glutamicumn complementing dapE of Escherichia coli," 40:3349-56 (1994) 2007203039 29 Jun 2007 2 (continued) X8206 16S rDNA l6S ribosomal RNA Ruimy, R. et al. "Phylogeny of the genus Corynebacterium deduced from I analy'ses of small-subunit ribosomal DINA sequences," Int. JS.. Bacteriol, 45(4):740-746 (1995) X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; Serebrijski, 1. et al. "Multicopy suppression by asd gene and osmotic stressdependent complementation by heterologous proA in proA mutants," J.

Bacteriol., 177(24):7255-7260 (1995) XR 92929 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.

177(24):7255-7260 (1995) X84257 16S rDNA 16S ribosomal RNA Pascual, C. et al. "Phylogenetic analysis of the genus Corynebacterium based 16S rRNA gene sequences," Int. J Sys. Bocteriol. 45(4):724-728 (1995) X985965 aroP; dapE Aromatic amino acid permease; Wehrmann et al. "Functional analysis of sequences adjacent to dapE of C.

glutamicum proline reveals the presence of aroP, which encodes the aromatic acid transporter," J. Bacteriol., 177(20):5991-5993 (1995) X86 157 argB; argC; argD; Acerylglutamnate kinase; N-acetyl-gamma- Sakanyan, V. et "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; omnithine steps of the arginine pathway," Microbiology, 142:99-108 (1996) carbamoyltransferase; glutamate Naceryltransferase X89084 pta; ackA Phosphate acetylItrans ferase; acetate kinase Reinsoheid, D.J. et al. "Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operen encoding ____________phosphotransacetylase and acetate kinase,"' Microbiology, 145:503-5 13 (1999) X99850 attB Attachment site Le Marrec, C. et al. "Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus C70," J. Bacierial., 178(7):1996-2004 (1996) X90356 Promoter fragment Fl Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90357 Promoter fragment F2 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90358 Promoter fragment F 10 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 FO3 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) 2007203039 29 Jun 2007 Table 2 (continued) IX90360 I rmtrfamn 2 aeN.e "Pro notcrs from Corynebacteriurn gluiaiiiicumi: ciuning, molecular analysis and search fr a consensus motif," Microbiology, (1996) X90361 Promoter fragment F34 Patek, M. et al- "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90362 Promoter fragment F37 Patek, M. et al. "Promoters from C. glutamicum: cloning, molecular analysis search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90363 Promoter fragment F45 Patek, M. et al. "Promoters from Corynebacterium glutamicum: 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 glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X- 90365 Promoter fragment P75 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X 9-0366 Promoter fragment PFI101 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90367 Promoter fragment PP 104 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology 142:1297-1309 (1996) X90368 Promoter fragment PP 109 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. fBi. Chem., (1996) X93514 betP Glycine betaine transport system Peter, IH. et al. "Isolation, characterization, and expression of thle Corynebacterium glutamicum betP gene, encoding the transport system for thle solute glycine betaine," J. lBacteriol., 178(1 7):5229-5234 (1996) X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of thle dapB-OR~F2dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes in L-lysine synthesis," Riotechnol. Lef., 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 Corynebaccerium glutamicum," Mo.

icrobiol, 22(5):815-826 (1996) 2007203039 29 Jun 2007 Table 2 (continued)I X96580 panB; panG; xylB 3-methyl-2-oxobutanoate Sahm, H. et al. "D-pantothenate synthesis in Gorynebacterium glutamicumn and hydroxymethyltransferase; pantoate-beta- use of panBC and genes encoding L-valine synthesis for D-pantothcnate alanine ligase; xylulokinase overproduction," App. Environ. Microbiol., 65(5):1973-1979 (1999) X96962 Insertion sequence IS 1207 and transposase XR99289 Elongation factor P Ramos, A. et al. "Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacteriumn lactoferrncntum (Corynebacterium glutamicum ATCC 13869)," Gene, 198:217-222 (1997) Y00140 thrB H-omoserine kinase Mateos, L.M. et al. "Nucleotide sequence of the homoserine kinase (thrB) gene of the Brevibacterium lactofermentum," Nucleic Acids Res., 15S(9):3922 (1987) Y00151 ddh Meso-d iam inopi me late D-dlehydrogenase Ishino, S. et al. "Nucleotidle sequence of the meso-diaminopimelate D- (EC 1.4.1.16) dlehydrogenase gene from Corynebacteriumn glutamicum," Nucleic Acids Res., 15(9):3917 (1987) Y00476 thrA Homoserine dlehydrogenase Mateos, L.M. et al. 'Nucleotidle sequence of the homoserine dlehydrogenase (thrA) gene of the Brevibacterium lactofermentum," Nucleic Acids Res., 15(24):10598 (1987) Y00546 horn; thrB Homoscrine dehydrogenase; homoserine Peoples, O.P. et al. "Nucleotide sequence and fine structural analysis of (he kinase Corynebacterium glutam icumn hom-thrB operon," Mci. Microbiol., 2(1 ):63 -72 (1988) 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.

protein; cell division protein Genet., 259(1):97-104 (1998) Y09163 putP High affinity proline transport system Peter, H. et al. "Isolation of the putP gene of Corynebacterium glutamnicumproline and characterization of a low-affinity uptake system for compatible solutes," Arch. Microbiol, 168(2): 143-151 (1997) Y09548 pyc Pyruvate carboxylase Peters-Wendisch, 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 IleuB gene from Corynebacterium App. Microbiol. Biolechnol., 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," Microbiol., 145:539-548 (1999) Y 12537 proP Proline/ectoine uptake system protein Peter, H. et al. "Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: Identification, sequenci .ng, and characterization of the proline/ectoine uptake system, ProP, and the ectoinelproline/glycine betaine carrier, EctP,"J. Bacteriol., 180(22):6005-6012 (1998) 2007203039 29 Jun 2007 2 (continued) Y1321 TlnA lutrn"esyrtheaseJakoby, 114. ct at. "Isolation. ofCoryriebacitiumj glutarnicumn ginA gene -encoding glutamime synthetase FEMS Microbiol Left., 154(1 1-88 (1997) Yl6642 lpd Dihydrolipoamide dehydrogenase Y18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis of the integration functions of φ304L: An integrase module among corynephages," Virology, 255(1): 150-159 (1999) Z21501 argS; lysA Arginyl-tRNA synthetase; diaminopimelate Oguiza, J.A. et al. "A gene encoding arginyl-tRNA synthetase is located in the decarboxylase (partial) upstream region of the lysA gene in Brevibacterium l *actofermentum: Regulation of argS-lysA cluster expression by arginine," J.

Bacteriol., 1 75(22):7356-7362 (1993) Z21502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et al. 'A cluster of three genes (dapA, orf2, and TdapB) of dihydrodipicolinate reductase Brevibacterium lactofermentumn encodes dlihydrodipicolinate reductase, and a third polypeptide of unknown function," J. Bacterial., 1 75(9):2743-2749 Z29563 thrC Tfhreonine synthase .Malumbres, M. et al. "Analysis and expression of the thrC gene of the encoded synthase," Appi. Environ. MicrobiaL., 60(7)2209-2219 (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 lactofermentum: Characterization of sigA and sigB," J. Bacterial., 178(2):550- 553 (1996) Z49823 galE; dtxR Catalytic activity UDP-galactose 4- Oguiza, J.A. et al "T'he galE gene encoding the UDP-galactose 4-epimerase of epimerase; diphthieria toxin regulatory Brevibacterium lactofermentum is coupled transcriptionally to the dmdR protein gene," Gene, 177:103-107 (1996) Z49824 orfl; sigB Sigl3 sigma factor Oguiza, J.A. et al "Multiple sigma factor genes in Brevibacterium lactoferrnentum: Characterization of sigA and sigB," J Bacterial, I178(2):550- 553 (1996) Z66534 Transposase Correia, A. et al. "Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentumn ATCC 13869," Gene, 170(l):9 1-94 (1996) A sequence for this gene was published in the indicated reference. However, the sequence obtained by the inventors of the present application is significantly longer than thie published version. It is believed that the published version relied on an incorrectstart codon, and thus represents only a fragment of the actual coding region: TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention Brevibacterium am moniagenes 21054 Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium ammoniagenes .19353 Brevibacterium ammoriiagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibactcriwn ammoniagenes 215S3 Brevibacterium *ammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brcvibacterium flavum 21129 Brcvibacterium flavum 21518 Brcvibacterium flavum BI 11474 Brevibacterium flavurn B 1472 Brevibacterium flavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brvibacterium flavum 21475 Brevibacterium flavum 21517 Brevibactcrium fiavum 21528 Brevibacterium fiavum 21529 Brevibacterium flavum B] 11477 Brevibacterium flavum BI 11478 Brevibacterium flavum 21127 Brevibacterium flavum BI 1474 Brevibacterium healii 15527 Brevibacterium ketoglutamicum 21004____ Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevibacterium lactofermnentum Brevibacterium lactofcrmentum 74 Brevibacterium lactoferrnennam 77 Brevibacterium lactofermentum 21798 Brevibacterium lactofermentum 21799 Brevibacterium lactofermnenrum 21800 Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B] 11470 Brevibacterium lactofermentum BI 11471 87 Geu. pce FERM 'NRRt ECr .,NCIM BS NCTC DSMZ; Brevibacterium lactofermentrm 21086 Brevibacterium lactofermennimn 21420 Brevibacterium lactofermentumn 21086 Brevibacterium lactoferynentum 31269 Brevibaccerium linens 9174 Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacterium paraffinolyticum 116 Brevibacterium Spec. 717.73 Brevibacterium Spec. _____717.73 Brevibacterium Spec. 14604 Brevibacterium Spec. 21860 Brevibacterium Spec. 21864 Brevibacterium Spec. 21865 Brevibacterium Spec. 21866 Brevibacterium Spec. 19240 Cornebacterium acetoacidophilum 21476 Cornebacterium acetoacidophilum 13870 Cormnebacterium acetoglutamicum BI 11473 Cot nebacterium acetoglutrmicumn B 11475 Corynebacterium acetoglutamnicumn 15806 Corenebacterium acetoglutrmicumn 21491 Coi-nebacterium acetoglutamicum 31270 Corynebacteriumn acetophilum _____B3671 Corynebacterium ammoniagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense 21496 Corinebacterium glutamnicumn 14067 Corynebacterium glutamnicumn 39137 Corynebacterium glutamnicumn 21254 Corynebacterium glutrmicumn 2155 Corynebacterium glutamnicumn 31830 Corynebacterium glutamnicumn 13032 Corynebacterium glutamnicumn 14305 Corynebacteriumn glutamnicumn 15455 Corynebacterium glutamnicumn 13058 Corynebacterium glutamnicumn 13059 Corynebacterium giutamnicumn 13060 Corynebacterium glutrmicum 21492 Corynebacterium glutamnicumn 21513 Corynebacterium glutrmicumn 21526 Corynebacteriuin glutrmicumn 21543 Corynebacterium glutamnicum 13287 Corynebacterium glutamnicumn 21851 Corynebacterium glutamnicumn 21253 Cory)nebacterium glutrmicumn 21514 Corynebacterium giutamnicumn 21516 Corynebacterium glutamicum 21299 88 e-jV-ER -7R C~ CB IX. Q- fDSM-Z- Corynebacterium glutarnicum 21300 Corynebacterium glutamicum 39684 Corynebacterium giutamicum 21488 Corynebacterium glutanmicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 19223 Corynehacterium glutamicum 13869 Corynebacterium glutamicum 21157 Corynebacterium gluta-micum 21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum 21355 Corynabacterium glutamicurn 31808 Corynebacterium glutamicum 21674 Coryncbacterium glutarnicum 21562 Corynebacterium glutamicum 21563 Corynebacterium glutamicum 21564 Corynebacterium giutamicum 21565 Corynebacterium giutamicum 21566 Corynebacterium glutamicum 21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum 21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum 21573 Corynebacterium glutarnicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 19050 Cor-ynebacterium glutamicumn 19051 Coi-ynebacterium glutamicum 19052 Corynebacterium glutamicumn 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutam icum 19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum 19185 Corynebacterium glutamicum 13286 Corync~bacterium glutarnicuM 21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum .21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum B8183 Corynebacterium glutamicum 8182 Corynebacterium giutainicum B12416 Corynebacterium glutamicum B12417 89 CorYnebacterium glutamicum B124]8 Corynebacterium glutainicum _BI 11476 Corynebacterium glutamicum 21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 3 1088 Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacterium spec. 3 1090 Corynebacterium spec. 3 1090 Cor-ynebacterium spec. 15954 20145 Corynebacterium spec. 21857 Corynebacterium spec. 21862 Corynebacterium spec. 21863 ATCC: American Type Culture Collection, Rockville, MD, USA FERM: Fermentation Research Institute, Chiba, Japan NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor Schimmelcultures, Baamn, NL NCTC: National Collection of Type Cultures, London, UK DSMZ: Deutsche Samm lung von Mikroorganismen und Zellkulturen, Braunschweig, Germnany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (4th edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.

2007203039 29 Jun 2007 Table 4: Alignment Results Length Accession Name of Genbank Hit ID 0 length Genbank Hit i,.I LU J Source of Genbank Hit %/homologiy Date of ICaPt) Denosit 37.148 13-Jul-99 rxaOO03 996 GBGSS4:A0713475 581 AQ713475 GB-HTG3:AC007420 130583 AC007420 GB-TG3:AC007420 130583 AC007420 ixa00014 903 GBBA1:MTCY3A2 25830 Z83867 HS_5402_B2_A12_T7A RPCI-i I Human Male BAG Library Homo sapiens genomic clone Plate=978 Col=24 Row=B, genomic survey sequence.

Drosophila melanogaster chromosome 2 clone BACRO7M1 0 (D630) RPCI-98 07.M.1 map 24A-24D strain y; cn bw sp, -SEQUENCING IN PROGRESS unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR07M1O (D630) RPCI-98 07.M.10 map 24A-24D strain y: cn bw sp, SEQUENCING IN PROGRESS-% 83 unordered pieces.

Homo sapiens D~rosophiia metanogaster 34,568 Drosophila meianogaster 34,568 20-Sep-99 20-Sep-99 GBBAI:MLCB1779 GBBA1:SAPURCLUS rxaOOO3O 513 GBEST2I:C89713 GBE5T28:AI497294 GBEST2I:C92167 rxaOOO32 1632 GB-BA2:AF010496 43254 9120 767 Z98271 X92429 C89713 Mycobacterium tuberculosis H37Rv compiete genome; segment 136/162. Mycobaclerlum tuberculosis Mycobacterium leprae cosmid 61779. Mycobacterium leprae Salbonigel napH, pur7. purlO. pure, pur4, pur5 and pur3 genes. Streptomyces anulatus C89713 Dictyostelium dlscoideum SS (H.Urushihara) Diclyostelium discoideum Dictyosteilum dlscoideum cDNA clone SSG229, mRNA sequence.

fb63g03.yl Zebrafish WashU MPIMG EST Danlo rerio cDNA 5' similar to Oanio rerio SW:AFP4_MYOOC P80961 ANTIFREEZE PROTEIN LS-12.:, mRNA sequence.

C92167 Dictyostelium discoideum SS (H.Urushihara) Dictyostelium discoideumn Dictyostelium discoideum cDNA clone SSD1 79, mRNA sequence.

Rhodobactor capsuiatus strain SBI003, partiai genome. Rhodobacter capsuiatus 58,140 17-Jun-98 484 A1497294 637 C92167 189370 AF010496 57,589 55.667 45,283 42,99 1 44,444 39,689 8-Aug-97 28-Feb-96 20-Apr-98 11 -MAR-1 999 12-Jul-99 12-MAY-i1998 22-OCT-i1997 1 6-Jul-98 GBBA2:AF018073 GBBA2:AF045245 EMPAT:E11760 GBPAT:126124 GBIN1:LMFL5883 9810 AF018073 Rhodobacter sphaeroldes operon regulator (smoG), peripiasmic sorbitol-binding Rhodobacter sphaeroides 48.045 protein (smoE), sorbitol/mannitol transport inner membrane protein (smoF), sorbitol/mannitol transport inner membrane protein (smoG), sorbitol/mannitol transport ATP-blndlng transport protein (smoK), sorbitol dehydrogenase (smoS), mannitol dehydrogenase (mtlK), and periplasmic mannilol-binding protein (smoM) genes, complete cds.

5930 AF045245 Kiebsielia pneumoniae D-arabinitol transporter (dalT), 0-arabinitol klnase (dalK), 0-arabintol dehydrogenase (dalD), and repressor (daIR) genes, complete cds.

6911 El 1760 Base sequence of sucrase gene.

Klebsieila pneumoniae Gorynebacterlum glutamicum 38,514 rxaOO041 1342 6911 126124 Sequence 4 from patent US 5556776. Unknown.

3 1934 ALI117384 Leishmania major Friedlin chromosome 23 cosmid L5883, complete sequence. Leishmania major 99.031 08-OCT-1997 (Ret. 52, Created) 99,031 07-OCT-1996 43,663 21-OCT-1999 94,767 08-OCT-1997 (Rel. 52, Created) 94,767 07-OCT-i1996 rxaOOO42 882 EM_PAT:.E1 1760 6911 El11760 Base sequence of sucrase gene.

6911 126124 Sequence 4 from patent US 5556776.

Gorynebacterium glulamicum GBPAT:126124 Unknown.

2007203039 29 Jun 2007 GBINI:CEU33051 nxa00043 1287 GB PAT:126124 EM_PAT:E1 1760 G8BPR:ACOO51 74 rxa00098 1743 GB-BA1:MSU88433 GBBA1:SC5A7 GB-BA1:MTCYIOD7 ra00148 2334 GBBA1:MTCY277 GBBAI:MSGY456 GB-BA1 :MSGY1 75 rxaOOl49 1971 GBBAI:M.SGY456 GBBA1:MTCY277 rxa00195 684 GBBA1:M7CY274 GBBA1:MSGB1529CS GBBA1:MTCY274 rxaOOI96 738 GBBAl:MTCY274 GBBA1:MTCY274

GBRO:RATCBRQ

rxa00202 1065 GB-ESTl I:AA253618 GBEST26:A1390284 4899 U33051 6911 126124 6911 E11760 39769 1928 40337 39800 38300 37316 18106 37316 18106 38300 39991 36985 39991 39991 39991 10752 313 490 467 44882 70287 AC0051 74 U88433 AL031 107 Z79700 Z79701 ADOOQOOlI AD000015

ADOOOOOI

AD000015S Z79701 Z74024 L78824 Z74024 Z74024 Z74024 M55532 AA25361 8 A1390284 A1390280 Z99263 AL02 1287 Table 4 (continued) Caenorthabditis etegans sur-2 mRNA. complete cds. Caenorhabditis etegans Sequence 4 from patent US 5556776. Unknown.

Base sequence of sucrase gene. Corynebacterium glutamicum Homo sapiens clone UWGC:g1564a012 from 7pl4-15, complete sequence. Homo sapiens Mycobacterium smegmatis phosphoglucose isomerase gene, complete cds. Mycobacterium smegmatis Streptomyces coelicolor cosmid 5A7. Streptomyces coeticolor Mycobacterlumn tuberculosis H37Rv complete genome: segment 44/162. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 65/1 62. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone y456. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone yl 75. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone y456. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone y175. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 65/1 62. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 126/162. Mycobacterium tuberculosis Mycobactedriu leprae cosmld B1529 DNA sequence. Mycobacterium leprae Mycobacterium tuberculosis H37Rv complete genome: segment 126/162. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome: segment 126/162. Mycobacterium tuberculosis Mycobacteriumn tuberculosis H37Rv complete genome; segment 126/162. Mycobacterium tuberculosis Rat carbohydrate binding receptor gene, complete cds. Rattus norvegicus mw95clO.rI Soares mouse NML Mus musculus cDNA clone IMAGE:678450 Mus musculus mRNA sequence.

mw98a03.yl Soaros mouse NML Mus musculus cONA clone IMAGE:678508 5 Mus muscutus similar to TR:009171 009171 BETAINE-HOMOCYSTEINE METHYLTRANSFERASE:, mRNA sequence.

mw95cIO.yl Scares mouse NMVL Mus musculus cONA clone IMAGE:678450 Mus musculus mRNA sequence.

Mycobacterium leprae cosmid 8637. Mycobacterium teprae Mycobacterium tuberculosis H37Rv complete genome; segment 132/162. Mycobacterium tuberculosis 40,276 97,591 97,591 35,879 62,658 37,638 36,784 67,457 40,883 67,457 35,883 5 1.001 51.001 35,735 57,014 41,892 41,841 36.599 36,212 38,816 42,239 37,307 58,312 36,632 23-Jan.96 07-OCT.1996 08-OCT-i1997 (Ret. 52, Created) 24-Jun-98 19-Apr-97 27-Jul-98 17-Jun-98 17-Jun-98 03-DEC-i1996 lo-DEC-1996 03-DEC-i1996 10-DEC.1996 17-Jun-98 19-Jun-98 15-Jun-96 19-Jun-98 19-Jun-98 19-Jun-98 27-Apr-93 13-MAR-1997 2-Feb-99 GBEST26:A1390280 GB BAI: MLCB637 GBBA1:MTVO12 2-Feb-99 17-Sep-97 23-Jun-99 rxaOO206 1161 2007203039 29 Jun 2007 GBBA1:SC6E1O rxa00224 1074 GBBA1:BJU32230 GBBA1 :PDEETFAB GBHTG3:AC009689 rxa00225 909 GBRO:AF060178 GBGSSII:A0325043 GBEST31:A1676413 Fxa00235 1398 GB-BA1:MTCYIOG2 GB BA2:AF061753 GBBA2:AF086791 rx00246 1158 GB_6A2:AF012550 GB-PAT:E03856 GBBA1:BACADH-T rxaOO251 831 GBBA1:MTCY2OG9 GBBA1:MTVOO4 GBBA1:MTVOO4 rxa00288 1134 GSBBA2:AF050114 GBGSS3:B16984 GB_1N2:AF144549 rxa00293 1035 GBEST1:T28483 23990 1769 2440 177954 2057 734 551 38970 37211 37867 2690 '1506 1688 37218 69350 69350 1038 469 7887 313 AL1 0966 1 U32230 1 14864 AC009689 AF060 178 AQ325043 A1676413 Z92539 AF061 753 AF086791 AF01 2550 E03856 090421 Z77 162 AL009 198 AL0091 98 AF0501 14 B16984 AF 144549 T28483 Table 4 (continued) Streptomyces coelicotor cosmid 6E10. Streptomyces coelicolor A3(2) Bradyrhizobium japonicum electron transfer flavoprotein small subunit (el(S) nd Bradyrhizobium japonicum large subunit (etf.) genes, complete cds.

Paracoccus denitrificans electron transfer flavoprotein alpha and beta subunit Paracoccus denitrificans genes, complete cds's.

Homo sapiens chromosome 4 clone 104F7 map 4, LOW-PASS SEQUENCE Homo sapiens

SAMPLING.

Mus musculus heparan sulfate 2-suifotransferase (Hs2st) mRNA, complete cds. Mus musculus mgxbOO2OJ0lr CUGI Rice Blast BAC Library Magnaporthe grisea genomic Magnaporthe grisea clone mgxbOO2OJ01lr. genomic survey sequence.

etmEST0l67 ElHi Elmeria tenella cDNA clone etmc074 mRNA sequence. Eimeria teneila Mycobacteriumn tuberculosis H37Rv complete genome; segment 47/152. Mycobacierium tuberculosis Nitrosomonas europaea CTP synthase (pyrG) gene, partial cds; and enotase Nitrosomonas europaea (eno) gene, complete cds.

Zymomonas mobtlis strain ZM4 clone 67E10 carbamnoylphosphate synthetase Zymomonas mobilis small subunit (carA), carbamnoyiphosphale synthelase large subunit (carB), transcription elongation factor (greA). enolase (eno). pyruvate dehydrogenase alpha subunit (pdhA), pyruvate dehydrogenase beta subunit (pdhB).

ribonuclease H (rnh), homoserine kinase homotog, alcohol dehydrogenase 11 (adhB), and excinuctease ABC subunit A (uvrA) genes, complete cds; and unknown genes.

Acinetobacter sp. BD413 ComP (comP) gene, complete cds. Acinetob *acter sp. BD413 gDNA encoding alcohol dehydrogenase. Bacillus stearothermophilus B.stearothermophitus adhT gene for alcohol dehydrogenase. Bacillus stearothermnophilus Mycobacterium tuberculosis H37Rv complete genome; segment 25/162. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 144/1 62. Mycobacteriumn tuberculosis Mycobacterium tuberculosis H37Rv complete genome: segment 144/1 62. Mycobacterium tuberculosis Pseudomonas sp. W7 alginate Iyase gene, complete cds. Pseudomonas sp. W7 344A14.TVC CIT978SKAI Homo sapiens genomic clone A-344A14, genomic Homo sapiens survey sequence.

Aedes albopictus ribosomal protein L34 (rp]34) gene, complete cds. Aedes albopictus EST46182 Human Kidney Homo sapiens cONA 3Send similar to flavin- Homo sapiens containing monooxygenase 1 (HT:1 956), mRNA sequence.

38.616 48,038 48. 35 1 38,756 39,506 38,333 35,542 65,759 58.941 61,239 53.726 51 .688 51,602 42,875 40,380 41,789 49,898 39,355 36,509 42,997 5-Aug.99 25-MAY-1 996 27-OCT-i1993 28-Aug-99 18-Jun-98 8-Jan-99 19-MAY-i1999 17-Jun-98 31 -Aug-98 4-Nov-98 27-Sep-99 29-Sep-97 7-Feb-99 17-Jun-98 18-Jun-98 18-Jun-98 03-MAR-i1999 4-Jun-98 3-Jun-99 6-Sep-95 2007203039 29 Jun 2007 2134 M64082 GBEST32:A1734238 512 A1734238 rxa00296 2967 GB-HTG6:AC0I 1069 168266 AC011069 GBEST15:AA531468 GB_HTG6:ACOI 1069 rxaOO310 558 GBVI:VMVY1678O

GB_VI:VARCG

GB_VI:WCGAA

rxa003i7 777 GBHTG3:AC009571 GBHTG3:AC009571 GBPR3:AC005697 rxa00327 507 GBBAI:LCATPASEB GBBA1:LCATPASEB rxa00328 615 GBBA1:STYPUTPE GB_BA1:STYPUTPF GBBAI :STYPUTPI rxa00329 1347 GBPR3:AC004691 GBPR4ACOO-4916 GBPR3:AC004691 rxaOO340 1269 GBBAi:MTCY427 GB-GSS1 2:AQ4 12290 GBPL2:AF1 12871 rxa00379 307 GBHTGI:CEY56A3 GBHTG1:CEY56A3 414 AA531468 168266 AC01 1069 166986 Y16780 186103 L22579 185578 X69198 159648 AC009571 15964 AC009571 174503 AC005697 1514 X64542 1514 X64542 1887 L01138 1887 L01139 1689 L01142 141990 AC004691 129014 AC004916 141990 AC004691 38110 Z70692 238 AQ412290 2394 AF112871 224746 AL022280 224746 AL022280 Table 4 (continued) Human flavin-containing monooxygenase (FMOi) mRNA, complete cds.

zb73c05.y5 SoaresjfetaljlungNbHL1 9W Homo sapiens cDNA clone IMAGE:309224 5' similar to gb:M6>4082 DIMETHYLANILINE MONOOXYGENASE (HUMAN);, mRNA sequence.

Drosophila melanogaster chromosome X clone BACRi 1H20 (D881) RPCI-98 111.1-1.20 map 12B-12C strain y; cn bw sp,- SEQUENCING IN PROGRESS -,92 unordered pieces.

nj63d12.sl NCI_-CGAP_PrlO Homo sapiens cONA clone IMAGE:997175, mRNA sequence.

Drosophila melanogaster chromosome X clone BACRI 11-20 (0881) RPCI-98 1 1.1-.20 map 12B-12C strain y; pn bw sp, -SEQUENCING IN PROGRESS ",92 unordered pieces.

variola minor virus complete genome.

VarlIola major virus (strain Bangladesh-i 975) complete genome.

Variola virus DNA complete genome.

Homo sapiens chromosome 4 clone 57_-A_-22 map 4, -SEQUENCING IN PROGRESS 8 unordered pieces.

Homo sapiens chromosome 4 clone 57_A_22 map 4, SEQUENCING IN PROGRESS 8 unordered pieces.

Homo sapiens chromosome 17, clone hRPK.138P22, complete sequence.

L.casei gene for ATPase beta-subunit.

L.casei gene for ATPase beta-subunit.

Salmonella (S2980) proline permease (pulP) gene. 5' end.

Salmonella (S2983) proline permease (pulP) gene, 5' end.

Salmonella (S3015) proline permease (pulP) gene. S'end.

Homo sapiens PAC clone DJ0740D02 from 7p14-p1 5. complete sequence.

Homo sapiens clone D.10691114, complete sequence.

Homo sapiens PAC clone DJ0740DO2 from 7p14-p15, complete sequence.

Mycobacterium tuberculosis H37Rv complete genome: segment 99/1 62.

RPCI-1 1-195H2.TV RPCi-1 1 Homo sapiens genomnic clone RPCi-1 1-1951-2.

genomic survey sequence.

Astasia longa small subunit ribosomal RNA gene, complete sequence.

Caenorhabditis elegans chromosome III clone Y56A3, SEQUENCING IN PROGRESS In unordered pieces.

Caenorhabditis elegans chromosome III clone Y56A3, SEQUENCING IN PROGRESS in unordered pieces.

Drosophila melanogaster 33,890 Homo sapiens Drosophila melanogaster variola minor virus Variola major virus Variola virus Homo sapiens Homo sapiens Homo sapiens Lactobacillus casel Lactobacillus casel Salmonella sp.

Salmonella sp.

Salmonella sp.

Homo sapiens Homo sapiens Homo sapiens Mycobacterium tuberculosis Homo sapiens Astasla longa Caenorhabditis elegans Caenorhabdilis elegans Homo sapiens Homo sapiens 37.915 8-Nov-94 41,502 14-Jun-99 40,821 30,963 35,883 34,664 36,000 36,988 36,988 36.340 34.664 39,308 39,623 39,623 42,906 38.142 38,549 35,865 38.94 0 36,555 36,465 35,179 35.179 02-DEC-i1999 20-Aug-97 02-DEC-i1999 2-Sep-99 12-Jan-95 1 3-DEC-i1996 29-Sep-99 29-Sep-99 09-OCT-i1998 il-DEC-i1992 1 1-DEC-1992 09-MAY-i1996 09-MAY-i1996 09-MAY- 1996 16-MAY-1998 17-Jul-99 16-MAY-i 998 24-Jun-99 23-MAR- 1999 28-Jun-99 6-Sep-99 6-Sep-99 2007203039 29 Jun 2007 rxaOO3Bl 729 GBPR2:HS134019 86897 GBGSS4:AQ730532 416 GB_EST23:AI-120939 561 Table 4 (continued) AL03,4555 Human DNA sequence from clone 134019 on chromosome I1p36.I1-36.33. Homo sapiens complete Bequence.

AQ730532 HS_2149_Al_C06_T7C CIT Approved Human Genomic Sperm Library D Homo sapiens Homo sapiens genomic clone Plate=2149 CoI=1 1 Row=-E, genomic survey sequence.

A1120939 ub74f05.rl Scares mouse mammary gland NMLMG Mus musculus cDNA clone Mus musculus IMAGE: 1383489 5 slmilar to gb:J04046 CALMODULIN (HUMAN); gb:M1 9381 Mouse calmodulin (MOUSE):, mRNA sequence.

A1120939 ub70f05.r0 Soares mouse mammary gland NMLMG Mus musculus cDNA clone Mus musculus IMAGE: 1383489 5'similar to gb:J04046 CALMODULIN (HUMAN): gb:M1 9381 Mouse calmodulin (MOUSE):, mRNA sequence.

40.604 35.766 23-Nov-99 15-Jul-99 41,113 2-Sep-98 41,113 2-Sep-98 GBEST23:A1120939 rxaOO385 362 GBEST32:A1726450 GDG554:A0740856

GB_PRI:HSPAIP

rxa00388 1134 GB_BAI:MVTY25DIO GBBA1:M5GY224 GB-HTG1:AP000471 rxa00427 909 GBBAl:M5GY126 GBBA1:MTY13DI2 GBHTG1:CEY48C3 rxa00483 1587 GBPR2:HSAFOOISSO GB-BA1 :LLCPJW565 GBHTG2:AC006754 rxaOO511 615 GB_PR3:HSE127C11 GBPR3:HSE127Cl1 rxa00512 718 GBBA1:MTCY22G8 561 565 A1726450 768 AQ740856 1587 X91809 40838 Z95558 40051 AD000004 72466 AP000471 37164 AD000012 37085 Z80343 270193 Z92855 173882 AF001550 12828 Y12736 206217 AC006754 38423 Z74581 38423 Z74581 22550 Z95585 BNLGHi5857 Six-day Cotton fiber Gossypium hirsutum cDNA 5' similar to Gossyplum hirs (AF0l 5913) SkblHs (Homo sapiens]. mRNA sequence.

HS_2274_A2_A07_T7C CIT Approved Human Genomic Sperm Library D Homo sapiens Homo sapiens genomic clone Plate=2274 Col=14 Row-A, genumic survey sequence.

H.sapiens mRNA for GAIP protein. Homo sapiens Mycobacterium tuberculosis H37Rv complete genome: segment 28/162. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone y2241. Mycobacterlum tuberculosis Homo sapiens chromosome 21 clone B2308H 15 map 2 1q22.3. Homo sapiens SEQUENCING IN PROGRESS-, In unordered pieces.

Mycobacterium tuberculosis sequence from clone y1 2 6. Mycobacteriumn tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 156/162. Mycobacterium tuberculosis Caenorhabditis elegans chromosome 11 clone Y48C3, SEQUENCING IN Caenorhabditis PROGRESS In unordered pieces.

Homo sapiens chromosome 16 BAG clone CIT987SK-334D1 1 complete Homo sapiens sequence.

Laclococcus lactis cremoris plasmid pJW565 DNA, abiiM, abiiR genes and Lactococcus Ia oriX. cremoris Caenorhabdltis elegans clone Y4011, SEQUENCING IN PROGRESS Caenorhabdilis unordered pieces.

Human DNA sequence from cosmid E127C1 1 on chromosome 22q1 1 .2-qter Homo sapiens contains STS.

Human DNA sequence from cosmid E127CI11 on chromosome 22q1 1.2-qter Homo sapiens contains STS.

Mycobacterium tuberculosis H-3 7Rv complete genome: segment 49/162. Mycobacterlurr utum elegans ctis subsp.

elegans 41,152 41.360 36,792 51,852 51,852 36,875 60,022 60,022 28,013 38.226 37.492 36,648 39.831 36.409 56,232 11-Jun-99 16-Jul-99 29-MAR-i1996 17-Jun-98 03-DEC.1996 1 3-Sep-99 10-DEC-i1996 17-Jun-98 29-MAY-i1999 22-Aug-97 01-MAR-i 999 23-Feb-99 23-Nov-99 23-Nov-99 17-Jun-98 tuberculosis 2007203039 29 Jun 2007 Table 4 (continued) M.smegmatis gILA gene for citrate synthase. G6B8i:MSULTA GBBA2:ECU73857 nxaOO517 1164 GB-HTG2:AC006911 GBHTG2:AC00691 1 GB-EST29:A1602 158 rxa00518 320 GBBA2:ECU73857 GBBA2:STU51879 40 rxa00606 2378 GB-EST32:AU068253 GBEST13:AA363046 GB-EST32:AU068253 rxa00635 1860 GBBA1:PAORF1 GBBA1:PAORF1 rxa00679 1389 GBPL2:AC010871 GB-PL1 :AT81 KBGEN GB-PL2:ACO1 0871 rxaOO680 441 GBPR3:AC004058 GBPLI:AT8IKBGEN GBPLI:AB026648 rxa00682 2022 GBHTG3:AC010325 GBHTG3:AC010325 GBPR4:AC008179 177b X60513 128824 U73857 298804 AC006911 298804 AC006911 481 A1602 158 128824 U73857 8371 U51879 12498 AE000140 376 AU068253 329 AA363046 376 AU068253 1440 X13378 1440 X13378 80381 AC010871 81493 X98130 80381 AC01 0871 38400 AC004058 81493 X98130 43481 A8026648 197110 AC010325 197110 AC010325 181745 AC008179 Mycobacterium smegmatis 56,143 20-Sep-91 Escherichia coli chromosome minutes 6-8. Escherichia coi Caenorhabditls elegans clone Y94H6x, SEQUENCING IN PROGRESS ~,Caenorhabditis etegans 115 unordered pieces.

Caenorhabdis elegans clone Y94H6x, SEQUENCING IN PROGRESS ~,Caenorhabditis etegans unordered pieces.

UI-R-ABO-vy-a-01-0-UI.s2 UI-R-ABO Rattus norvegicus cDNA clone UI-R-AB0- Rattus norvegicus vy-a-01-0-UI mRNA sequence.

Escherichla coti chromosome minutes 6-8. Escherichia coi Salmonella typhimurium proplonate catabolism operon: RpoN activator protein Salmonella typhimuuium homolog (prpR), carboxyphosphonoenolpyruvate phosphonomnutase homolog (prpB), citrate synthase homolog (prpC). prpD and prpE genes, complete cdls.

Escherichla col K-12 MG1655 section 30 of 400 of the complete genome. Escherichia coi AU068253 Rice callus Oryza sativa cDNA clone C 1 2658_9A, mRNA sequence. Oryza sativa EST72922 Ovary 11 Homo sapiens cONA 5' end. mRNA sequence. Homo sapiens AU068253 Rice callus Oryza sativa cONA clone 012658_9A, mRNA sequence. Oryza saliva Pseudomonas amyloderamosa DNA for ORF 1. Pseudomonas amyloderamosa Pseudomonas amylodleramosa DNA for ORF 1. Pseudomonas amytoderamosa Arabidopsis thallana chromosome III BAG T1601 1 genomic sequence, Arabidopsis thaliana complte sequence.

Athaliana 81lkbgenomic sequence. Arabidopsis thaliana Arabidopsis thaliana chromosome III BAC T16011 genomlc sequence, Arabidopsis thaliana complete sequence.

H-omo sapiens chromosome 4 clone B241P 19 map 4q25, complete sequence. Homo sapiens Athaliana 81kb genomic sequence. Arabidopsis thaliana Arabidopsis thaliana genomic DNA. chromosome 3, P1 clone: MU 15. complete Ara bidopsIs thaliana sequence.

Homo sapiens chromosome 19 clone CITB-E1-2568A17, SEQUENCING IN Homo sapiens PROGRESS 40 unordered pieces.

Homo sapiens chromosome 19 clone CITB-El_2568A17, SEQUENCING IN Homo sapiens PROGRESS 40 unordered pieces.

Homo sapiens clone NHO576FO1. complete sequence. Homo sapiens 48.563 14-Jul-99 37,889 37,889 40,833 49,688 50,313 49,688 41,333 34,347 4 1,899 53,912 54,422 38,244 36.091 37,135 36.,165 38,732 38,732 37,976 37,976 37.143 24-Feb-99 24-Feb-99 21-Apr-99 14-Jul-99 5-Aug-99 12-Nov-98 7-Jun-99 21-Apr-97 7-Jun-99 14-Jul-95 14-Jul-95 13-Nov-99 12-MAR-i 997 13-Nov-99 30-Sep-98 12-MAR-1 997 07-MAY-i1999 15-Sep-99 15-Sep-99 28-Sep-99 2007203039 29 Jun 2007 rxa00683 1215 GBBA2:AE000896 GBINI:OMBR7A4 GBEST35:AV163010 rxa00686 927 GB-HTG2:HSDJ137K2 GBHTG2:H50J137K2 GBESTI2:AA284399 rxaOO700 927 GBEST34:A1785570 GBEST25:A1256147 GBBA1:CARCGI2 rxa00703 2409 GBBA1:SC7H2 GBBA1:MTCY274 GBBA2:REU60056 rxa00705 1038 GBGSS1S:AQ604477 10707 212734 273 190223 190223 431 454 AE000896 AL1 09630 AV16 30 10 ALD49820 AL049820 AA284 399 A1785570 Table 4 (continued) Methanobacerium therrnoautotrophicumn from bases 1189349 to 1200055 Methanobacterium (section 102 of148) of the complete genome. thermoautotrophicum Drosophila nielanogaster clone BACR7A4. Drosophila melanogaster AV1 63010 Mus musculus head C578U6J 13-day embryo Mus musculus cONA Mus musculus clone 3110006J22, mRNA sequence.

H-omo sapiens chromosome 6 clone RPI-137K2 map q25.1-25.3, Homo sapiens SEQUENCING IN PROGRESS ',In unordered pieces.

Homo sapiens chromosome 6 clone RP1-137K2 map q25.1-25.3, Homo sapiens SEQUENCING IN PROGRESS in unordered pieces.

zs57b04.rl NCItOGAP_GCBI Homo sapiens cDNA clone IMAGE:701551 5% Homo sapiens niRNA sequence.

uj44d03.xl Sugano mouse liver mlia Mus musculus cIDNA clone Mus musculus IMAGE:1922789 3 similar to gb:Z2B407 60S RIBOSOMAL PROTEIN L8 (HUMAN);, mRNA sequence.

u195el12.xl Sugano mouse liver nilia Mus musculus cDNA clone Mus musculus IMAGE:1890190 3'sImilar to gb:Z28407 60S RIBOSOMAL PROTEIN L8 (HUMAN):, mRNA sequence.

C. aurantlacus reaction center genes I and 2. Chloroflexus aurantiacus Streptomyces coelicotor cosmid 71-2. Streptomyces coeticolor A3(2) Mycobacterium tuberculosis H37Rv complete genome; segment 126/1 62. Mycobacterium 38.429 36.4 54 4 1,758 38,031 38,031 39.205 41,943 15-Nov-97 30-Jul-99 8-Jul-99 03-DEC-1 999 03-DEC-1 999 14-Aug-97 2-Jul-99 684 A1256147 40.791 12-Nov-98 2079 42655 39991 2520 505 rxa00782 1005 rxa00783 1395 rxa00794 1128 GB_EST1 I:AA224340 443 GB-EST5:N30648 291 GBBA1:MTCY1007 39800 GBBA1:MLCL373 37304 GBBA2:AF128399 2842 GBHTG2:AC008158 118792 GB-HTG2:AC008158 118792 GBPR3:AC005017 137176 GB-BA1:MTVO17 67200 X14979 AL1 09732 Z74024 U60056 A0604477 AA224340 N30648 Z79700 AL035500 AF128399 AC0081 58 AC0081 58 AC005017 AL021 897 tuberculosis Ratstonla eutropha formate de hydroge nase -like protein (cbb8c) gene, complete Ralstonia eutropha cds.

HS_72116_BiGO7-MR CIT Approved Human Genomlc Sperm Library D H-omo Homo sapiens sapiens genomic clone Plate=2l 16 Col=13 Row=N, genomic survey sequence.

zrl4eO7.sl Stratagene hNT neuron (#937233) Homo sapiens clDNA clone Homo sapiens IMAGE:648804 mRNA sequence.

yw77b02.sl Soaresjlacenta8to9weeks2NbHPIo9W Homo sapiens cDNA Homo sapiens clone IMAGE:258219 mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 44/162. Mycobacterium tuberculosis Mycobacterium Ieprae cosmid L373. Mycobacterium lepr Pseudomonas aeruginosa succinyl-CoA synthetase beta subunit (sucC) and Pseudomonas aeru succinyl-CoA synthetase alpha subunit (sucO) genes, complete cds.

Homo sapiens chromosome 17 clone hRPK.42.j_20 map 17, Homo sapiens SEQUENCING IN PROGRESS 14 unordered pieces.

Homo sapiens chromosome 17 clone hRPK.42_F_20 map 17, Homo sapiens SEQUENCING IN PROGRESS 14 unordered pieces.

Homo sapiens BAG clone GS214N13 from 7p14-p15, complete sequence. Homo sapiens Mycobacterium tuberculosis H37Rv complete genome: segment 48/162. Mycobacterium tuberculosis ae ginosa 37,721 56,646 37,369 51,087 39,617 35.129 43,986 63,327 62,300 53,698 35,135 35,135 35,864 40,331 23-Apr-91 2-Aug-99 19-Jun-98 16-OCT-i1996 10-Jun-99 11 -MAR-1 998 5-Jan-96 17-Jun-98 27-Aug-99 25-MAR-i1999 28-Jul-99 28-Jul-99 8-Aug-98 24-Jun-99 2007203039 29 Jun 2007 GB BAI:MLCB1222 G13PR_2:HS1 51614 Table 4 (continued) 34714 AL049491 Mycobacterium ieprae cosmid 13222.

128942 Z82 188 Human DNA sequence from clone 151B314 on chromosome 22 Contains rna00799 1767 rxa00800 1227 rxa00825 1056 GBPL2:AF016327 616 AF016327 GB-HTG2:HSDJ3lgM7 128208 AL079341 GB-HTG2:HS0J319M7 128208 AL079341 GB-BAI:MTV022 13025 AL021925 GBBAI:A8019513 4417 AB01 9513 GB3_PL1:SCSFAARP 7008 X68020 G13_6A1:MTY15C10 33050 Z95436 GBBAI:MLC62548 38916 AL023093 GB-BA2:AF169031 1141 AF169031 SOMATOSTATIN RECEPTOR TYPE 3 (SS3R) genepseudogene similar to ribosomal protein L39,RAC2 (RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 2 (P21-RAC2)) gene ESTs, STSs. GSSs and CpG Islands.

complete sequence.

Hordeum vulgare Barpermi (permi) mRNA. partial cds.

Homo sapiens chromosome 6 clone RP1 -31 9M7 map p21.1-21.3.

SEQUENCING IN PROGRESS In unordered pieces.

Homo sapiens chromosome 6 clone RP1 -31 9M7 map p21.1-21.3, SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome: segment 1001162.

Streptomyces coelicolor genes for alcohol dehydrogenase and ABC transporter. complete cds.

S.cerevisiae SFA and ARP genes.

Mycobacterium tuberculosis H37Rv complete genome: segment 1541162.

Mycobacterlum Ieprae cosmid 82548.

Xanthomonas oryzae pv. oryzae putative sugar nucleotide epimeraseldehydratase gene, partial cds.

Mycobaciefium ieprae Homo sapiens Hordeum vulgare Homo sapiens Homo sapiens Mycobacterium tuberculosis Sireptomyces coelicolor Saccharomyces cerevisiae Mycobacterium tuberculosis Mycobacterium leprae Xanthomonas oryzae pv.

oryzae Caenorhabditis elegans Homo sapiens Homo sapiens Mycobacterium tuberculosis Candida dubliniensis Candida albicans Rhodobacter capsulatus Sinorhizobium meliloti Paralichthys olivaceus Homo sapiens 41,311 36,845 36,845 63.101 41,312 36,288 39,980 39,435 46.232 01-OCT-i1997 30-Nov-99 30-Nov-99 17-Jun-98 13-Nov-98 29-Nov-94 17-Jun-98 27-Aug-99 14-Sep-99 61,170 27-Aug-99 37.455 16-Jun-99 rxa00871 rxa00872 1077 GB_INI:CEF23HI2 GB-HTG2:AC007263 GBHTG2:AC007263 rxa00879 2241 GBBA1:MTVO49 GBPL2:C0U236897 GBPL1:CAACTIA rxa00909 955 GBBA2:AF010496 GBBA1 :RMPHA GB_EST16:C23528 rxa009l3 2118 GB-HTG2:AC007734 35564 167390 167390 40360 1827 3206 189370 7888 317 Z74472 AC007263 AC007263 AL022021 AJ236897 X16377 AF010496 X93358 C23528 Caenorhabditis elegans cosmid F23H-112, complete sequence.

Homo sapiens chromosome 14 clone BAC 79J20 map 14q31, SEQUENCING IN PROGRESS-, 5 ordered pieces.

H-omo sapiens chromosome 14 clone BAC 79J20 map 14q31.

SEQUENCING IN PROGRESS 5 ordered pieces.

Mycobacterium tuberculosis H37Rv complete genome; segment 81/162.

Candida dubliniensis AClI gene. exons 1-2.

Candida albicans acti gene for actin.

Rhodobacter capsulatus strain S81003, partial genome.

Rhizoblum meliloti pha[A,BC,D,E,F,G] genes.

C23528 Japanese flounder spleen Paralichihys olivaceus cONA clone HB5(2).

mRNA sequence.

Homo sapiens chromosome 18 clone hRPK.44_0 -1 map 18, SEQUENCING IN PROGRESS 18 unordered pieces.

34,502 35,714 35,714 36,981 38,716 36,610 51,586 48,367 41,640 34,457 08-OCT-i1999 24-MAY-i1999 24-MAY-i 999 19-Jun-98 1-Sep-99 1 0.Apr-93 1 2-MAY-i1998 1 2-MAR-1999 28-Sep-99 5-Jun-99 188267 AC007734 2007203039 29 Jun 2007 GBHTG2:A0007734 GBESTi 8:MA709478 rxa00945 1095 GBHTG4:ACO1O35I GBHTG4:AC010351 GBBA1:MTCYO5A6 188267 AC007734 406 AA709478 220710 AC010351 220710 AC010351 36631 Z96072 Table 4 (continued) H-omo sapiens chromosome 18 clone hRPK.440 map 18, SEQUENCING IN PROGRESS 18 unordered pieces.

vv34a05.rl Stratagene mouse heart (#937316) Mus musculus cDNA clone IMAGE: 1224272 mRNA sequence.

Homo sapiens chromosome 5 clone CITB-Hl_202286, -SEQUENCING IN PROGRESS 68 unordered pieces.

Homo sapiens chromosome 5 clone CITB-H1_2022BB, SEQUENCING IN PROGRESS 68 unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome; segment 120/162.

Homo sapiens Mus musculus Homo sapiens Homo sapiens Mycobacterium tuberculosis 34.457 42,065 36,448 36,448 36.2 18 5-Jun-99 24-DEC-1 997 31-OCT.1999 31-OCT-1999 17-Jun-98 rxa00965 rxa00999 1575 GBPAT:E13660 GBBAI:MTCY359 GBBA1:MLCB1788 rxa0l0lS 442 GBBA1:M1VOO8 GBBA1:MTVOO8 1916 36021 39228 63033 63033 rxaOlO2S 1119 GBBA1:SC7A1 32039 GBBA1:MSG81I723CS 38477 GBBAI:MLCB637 44882 rxaOlO4B 1347 GBBA2:AF017444 3067 GBBA1:BSUBOO13 218470 GBVL:HSV2HG52 154746 rxaOIO49 1605 GBHTG2:AC00251 8 '131855 GBHTG2:AC00251 8 131855 GBHTG2:AC00251 8 131855 rxaOlO77 1494 GB-PR3:H-15J653C5 85237 GBBA1:ECU29579 72221 GBBA1:ECU29579 72221 rxa0lO89 873 GBGSSB:A0044021 387 E13660 Z83859 AL0086D9 AL02 1246 AL02 1246 AL034447 L78825 Z99263 AF01 7444 Z99 116 Z86099 AC002518 AC00251 8 AC00251 8 AL049743 U29579 U29579 AQ044021 gONA encoding 6-phosphogiuconale dehydrogenase.

Mycobacterium tubercutosis H37Rv complete genome; segment 84/162.

Mycobaclerium icprae cosmid 81788.

Mycobacterium tuberculosis H37Rv complete genome; segment 108/162.

Mycobacterium tuberculosis H37Rv complete genome; segment 108/1162.

Streptomyces coelicolor cosmid WA.

Mycobacterium leprae cosmid 81 723 DNA sequence.

Mycobacterium ieprae cosmid 8637.

Slnorhizoblumn meiloti NAOP-dependent malic enzyme (tine) gene, complete cds.

Baciilus subtIlls compiete genome (section 13 of 21): from 2395261 to 2613730.

Herpes simplex virus type 2 (strain HG52). complete genome.

Homo sapiens chromosome X cione bWVXD2O, SEQUENCING IN PROGRESS 11I unordered pieces.

Homo sapiens chromosome X clone bWXD2O, SEQUENCING IN PROGRESS 11 un~rdered pieces.

Homo sapiens chromosome X clone bWXD2, SEQUENCING IN PROGRESS 11 unordered pieces.

Human DNA sequence from clone 653C5 on chromosome Ip 2 l.3-22.3 Contains CA repeatCDl S435), STSs and GSSs, complete sequence.

Escherichia coli K-1 2 genome: approximately 6110o62 minutes.

Escherichia coli K-1 2 genome; approximately 61 to 62 minutes.

CIT-HSP-2318C1 8.TR CIT-HSP Homo sapiens genomic clone 2318C18, genomic survey sequence.

Corynebacterium glutamicum Mycobacterium tuberculosis Mycobacterium leprae Mycobacterium tuberculosis Mycobacterium tuberculosis Streptomyces coelicolor Mycobacterium Ieprae Mycobacterium leprae Sinorhlzobium metiloti Bacillus subtilis human herpesvirus 2 Homo sapiens Homo sapiens Homo sapiens Homo sapiens Escherichia coli Escherchia coli Homo sapiens 98,349 38,520 64,355 39,860 39,120 55,287 56,847 56,676 53.660 37.255 38.08 1 35,647 35,647 26,180 36,462 41,808 36, 130 36,528 24-Jun-98 17-Jun-98 12 7 -Aug -99 17-Jun-98 1 5-DEC-1998 15-Jun-96 17-Sep-97 2-Nov-97 26-Nov-97 04-DEC-1998 2-Sep-97 2-Sep-97 2-Sep-97 23-Nov-99 1-Jul-95 1-Jul-95 14-Jul-98 2007203039 29 Jun 2007 Table 4 (continued) AQ042907 CIT-HSP-2318DI7.TR CIT-HSP Homo sapiens genomic clone 2318D17, GBGSS8:A0042907 392 GB-GSS8:AO04402i rxa0i093 1554 GB_BAi:CORPYKI GBBA1:MTCYOiB2 GBBA1:M1U65430 rxa01099 948 GBBA2:AF045998 GBBA2:AF051846 GB-GSSI :FR0005503 rxa0llil 5411 GBPR3:AC004063 GB_PR3:HS1 178121 GBHTG3:AC009301 rxaOii3O 687 GBHTG3:AC009444 GBHTG3AC009444 GB lNl:DMC66Ai rxa0ll93 1572 GB_BA1:CGASO19 EMPAT:E09634 GBBAi:MLU15iB6 rxa~l194 495 EMPAT:E09634 GB_BA1:CGASO19 GB_VI:HEPCRE4B rxa0l 200 387 2795 35938 1439 780 738 619 177014 62268 163369 164587 164587 34127 1452 1452 36241 1452 A0044021 L27126 Z95554 UJ65430 AF045998 AF051846 Z89313 AC004063 AL1 09852 AC009301 AC009444 AC009444 AL031 227 X76875 1109634 U 15186 E09634 genomic survey sequence.

CIT-H-SP-2318C18.TR CIT-HSP Homo sapiens genomic clone 2318018.

genomic survey sequence.

Corynebacterium pyruvale kinase gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 72/162.

Mycobacterium intracelfulare pyruvate kinase (pylrF) gene, complete cds.

Corynebactenium glutamicum inositol monophosphate phosphatase (impA) gene, complete cds.

Corynebactedrlu glutamicum phosphoribosylformlmino-5-amno-lphosphoribosyl-4- lmidazolecarboxamidle Isomerase (hisA) gene, complete cds.

Frubrlpes GSS sequence, clone 079B16aE8, genomic survey sequence.

Homo sapiens chromosome 4 clone B3218, complete sequence.

Human DNA sequence from clone RP5-1 178121 on chromosome X, complete sequence.

Homo sapiens clone NH0062F14, SEQUENCING IN PROGRESS *,5 unordered pieces.

Homo sapiens clone 1_0_3, SEQUENCING IN PROGRESS ~,8 unordered pieces.

Homo sapiens clone 1_0_3, SEQUENCING IN PROGRESS ,8 unordered piece s.

Drosophila melanogaster cosmid 66A1.

C.glutamicum (ASO 19) ATPase beta-subunit gene.

Brevibacterium flavumn UncD gene whose gene product Is Involved in Mycobacterium leprae cosmid L471.

Brevibacterium flavurn UncO gene whose gene product is involved in Oglutamicum (ASO 19) ATPase beta-subunit gene.

Hepatitis C genomic RNA for putative envelope protein (RE4B Isolate).

Homo sapiens Homo sapiens Corynebacterium glutarnicum Mycobacterium tuberculosis Mycobacterium intracellulare Corynebacterium glutamicum Corynebacterium glutamicum Fugu rubripes Homo sapiens Homo saplens Homo sapiens Homo sapiens Homo sapiens Drosophila melanogaster Corynebacterlum glutamicum Corynebacterium glutamicum Mycobacterium leprae Corynebacterium glutamicum Corynebacterium glutamicum Hepatitis C virus 35,969 44.5,45 100.000 63.771 67.061 99,615 100,000 37,785 35,835 37,873 37,240 38,416 38,416 38,416 99,931 99,242 39,153 100,000 100,000 36,769 14-Jul-98 14-Jul-98 07-DEC-i 994 17-Jun-98 23-DEC-i1996 19-Feb-98 12-MAR-1 998 01-MAR-1997 10-Jul-98 01-DEC-i1999 13-Aug-99 22-Aug-99 22-Aug-99 05-OCT-1998 27-OCT-i1994 07-OCT-i 997 (Rel. 52, Created) 09-MAR-i1995 07-OCT-I 997 (Rel. 52, Created) 27-OCT-1994 5-Apr-92 1452 X76875 414 X60570 2007203039 29 Jun 2007 n~a01201 1764 GBBAI:SLATPSYNA GBBAI:MTCY373 GB-BA1:MLU15186 rxa01202 1098 GBBAI:SLATPSYNA

GBBAI:SLATPSYNA

GBBA1:MCSOSSHC rxa0l204 933 GBPLI:AP000423 GB-HTG6:AC009762 GBHTG6:AC009762 rxa01216 1124 GBBA1:MTCY1OG2 GBBA2:AF017435 GBBAl :CCRFLBDBA rxa01225 1563 GB-BA2:AF058302 GB-HTG3:AC007301 GBHTG3:AC007301 rxa01227 444 GB-BA1:SERFDXA GB-BA1 :MSGY348 rxa0 1242 900 GBPR3:AC005697 GBt-HTG3:AC010722 G8BHTG3:AC0I 0722 8560 35516 36241 8560 8560 5538 154478 164070 184070 38970 4301 4424 25306 165741 Z22606 Z73419 U 15186 Z22606 Z22606 Y09978 AP000423 AC009762 AC009762 Z92539 AF0 17435 M69228 AF058302 AC007301 Table 4 (continued) S.lividans i protein and ATP synthase genes. Streptomyces lividans 66.269 Mycobacterium tuberculosis H-37Rv complete genome; segment 57/162. Mycobacterium 65,437 tuberculosis Mycobacterium Ieprae cosmid 1-471. Mycobacterium leprae 39,302 Slividans i protein and ATP synthase genes. Streptomyces lividans 57,087 Slividans i protein and ATP synthase genes. Streptomyces lividans 38,298 M.capsulalus orbx, orly, orfz, sqs and shc genes. Methylococcus capsulatus 37,626 Arabidopsis thaliana chtoroplast genomic DNA. complete sequence. Chloroptast Arabidopsis 38,395 strain:Columbia. thatiana Homo sapiens cdone RP1 1-114116, SEQUENCING IN PROGRESS ~,39 Homo sapiens 35,459 unordered pieces.

H-omo sapiens clone RP1 1- 1416~ SEQUENCING IN PROGRESS ,39 Homo sapiens 36,117 unordered pieces.

Mycobacterlum tuberculosis H37Rv complete genome; segment 47/162. Mycobacterium 39,064 tuberculosis Methylobacterlum extorquens methanol oxidation genes. g~mU-like gene, Methylobacterium 42,671 partial cds, and orfl-2, orfl-1, oriR genes, complete cds. extorquens C.crescenlus flagetlar gene promoter region. Caulobacter crescentus 41,054 Streptomyces foseofulvus trenolicin biosynthetic gene cluster, complete Streptomyces roseofulvus 36,205 sequence.

Drosophila metanogaster chromosome 2 clone BACRO4BO09 (13576) RPCI-98 Drosophila melanogaster 39,922 04.B.9 map 43E1 2-44F1 strain y; cn bw sp, -SEQUENCING IN PROGRESS 150 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACRO4BO09 (D3576) RPCI-98 Drosophila melanogaster 39,922 04.B.9 map 43E12-44F1 straIn y: cn bw sp, -SEQUENCING IN PROGRESS ',150 unordered pieces.

Saccharopotyspora er-ythraea ferredoxin (fdxA) gene, complete cds. Saccharopotyspora 64,908 erythraea Mycobacterium tuberculosis H37Rv complete genome; segment 511162. Mycobacteriumn 62,838 tuberculosis Mycobacterium tuberculosis sequence from clone y348. Mycobacterium -61,712 tuberculosis H-omo sapiens chromosome 17, clone hRPK.138P22, complete sequence. Homo sapiens 35,373 Homo sapiens clone NH0122L-09. SEQUENCING IN PROGRESS Homo sapiens 39,863 unordered pieces.

Homo sapiens clone NH-0122L-09, -SEQUENCING IN PROGRESS ,2 Homo sapiens 39,863 unordered pieces.

01-MAY-i995 17-Jun-98 09-MAR-i1995 01-MAY-i1995 01-MAY-1995 26-MAY-1998 15-Sep-99 04-DEC-1999 04-DEC- 1999 17-Jun-98 10-MAR-1998 26-Apr-93 2-Jun-98 17-Aug-99 11-Aug-99 13-MAR-I1996 17-Jun-98 10-DEC-1996 09-OCT-i1998 25-Sep-99 25-Sep-99 165741 AC007301 3869 37840 40056 174503 160723 160723 M61119 AL010186 AD000020 AC005697 AC01 0722 AC01 0722 2007203039 29 Jun 2007 rxa01243 1083 GBGSS10A025fl57 593 GB-INI1:CEKO5D4 GDIN1:CEKO5D4 rxa01259 981 GB-BAI:CGLPD GBHTG4:AC01C567 GBHTG4:AC010567 rxa01262 1284 GBBA2:AF172324 GBBA2:ECU78086 GBBAl:090841 rxa0l311 870 GB-PR3:AC004103 GBHTG3:AC007383 GBHTG3:AC007383 rxa0l3l2 2142 GB-BA2:AE000487 GB BA1:MTVOI6 GB-BA1 :U00022 rxa01325 795 GBHTG4:AC009245 GB-HTG4:AC009245 GBHTG4AC009245 rxaol 332 576 GS HTG6:ACOO7I 86 GBHTG6:AC007147 19000 19000 1800 143287 143287 14263 4759 20226 144368 215529 215529 13889 53662 36411 215767 215767 215767 225851 Table 4 (continued) AQ255057 rnYhnnnAR'Nni, CIO! Rice Blast BAC Library M agnzpcr~hc grsagcnomic 1M1agnaporthe grisea clone mgxbOOO8N0lr. genomic survey sequence.

Z92804 Caenorhabditis elegans cosmid K0504, complete sequence. Caenorhabdilis elegans Z92804 Caenorhabditis elegans cosmid K05D4, complete sequence. Caenorhabditis elegans Y16642 Coi-ynebacterium glutamicum lpd gene, complete 005. Corynebacteilum giutamicum AC010567 Drosophila melanogaster chromosome 3LJ69C1 clone RPCI98-1 1N6. Drosophila melanogaster SEQUENCING IN PROGRESS 70 unordered pieces.

AC01 0567 Drosophila melanogaster chromosome 31J69CII clone RPC198-1 1 N6, Drosophila melanogaster -SEQUENCING IN PROGRESS 70 unordered pieces.

AF172324 Escherichla coil GafF (gaIF) gene, partial cds; 0-antigen repeat unit transporter Escherichia coli Wzx (wzx), WbnA (wbnA), 0-antigen polymerase Wzy (wzy), WbnB (wbnB), WbnC (wbnC), WbnD (wbno), VWbnE (wbnE), UDP-Gic-4-epimerase GalE (galE), 6-phosphogluconate dehydrogenase Gnd (gnd), UDP-Gtc-6dehydrogenase Ugd (ugd). and WvbnF (wbnF) genes, complete cds: and chain length determinant Wzz (wzz) gene, partial cds.

U78086 Escherlchia col hypothetical uridine-5'-diphosphoglucose dehydrogenase (ugd) Escherlchla coli and 0-chain length regulator (wzz) genes, complete cds.

D90841 E.coli genomic DNA, Kohara clone #351(45.1-45.5 min.). Escherichia coil 35,448 35.694 100,000 37.178 37,178 59.719 23-OCT-1998 23-Nov-98 23-Nov-98 1 -Feb-99 16-00T-1999 16-OCT- 1999 29-OCT-i1999 59,735 5-Nov-97 AC004 103 AC007383 AC007383 AE000487 AL021841 U00022 AC009245 AC009245 AC009245 AC0071 86 Homo sapiens Xp22 BAC GS-619J13 (Genome Systems Human BAO library) complete sequence.

Homno sapiens clone NHO31IOK1 5. SEQUENCING IN PROGRESS ~,4 unordered pieces, Homo sapiens clone NH0310K1 5, SEQUENCING IN PROGRESS ',4 unordered pieces, Escherichia coil K-12 MG 1655 section 377 of 400 of the complete genome.

Mycobacterium tuberculosis. H37Rv complete genome; segment 143/162.

Mycobacterium leprae cosmid 1-308.

Homo sapiens chromosome 7, SEQUENCING IN PROGRESS 24 unordered pieces, Homo sapiens chromosome 7, SEQUENCING IN PROGRESS .24 unordered pieces.

Homo sapiens chromosome 7, SEQUENCING IN PROGRESS ~,24 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACRO3006 (0569) RPCI-98 03.D.6 map 32A-32A strain y; cn bw sp, SEQUENCING IN PROGRESS-, 91 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR19N18 (D572) RPCI-98 19.N.18 map 32A-32A strain y; cri bw sp, -SEQUENCING IN PROGRESS 22 unordered pieces.

Homo sapiens Homo sapiens Homo sapiens Escherlchla coli Mycobacterium tuberculosis Mycobacterium leprae Homo sapiens Homo sapiens Homo sapiens Drosophila melanogaster Drosophila melanogaster 37,904 37,340 36,385 36.385 39,494 46,252 46,368 36.016 36.0 16 39,618 35.366 35.366 21-MAR-1997 18-Apr-98 25-Sep-99 25-Sep-99 12-Nov-98 23-Jun-99 01-MAR-1994 2-Nov-99 2-Nov-99 2-Nov-99 07-DEC-i1999 07-DEC-1999 202291 AC007147 2007203039 29 Jun 2007 Table 4 (continued) Homo sapiens clone RPCI 11-375120. "SEQUENCING IN PROGRESS-, 25 Homo sapiens GB-HTG3:AC010207 207890 AC010207 unordcred picces.

1107 rxa0l365 1497 rxa01369 1305 rxa01377 1209 rxa01392 1200 rxa01436 1314 GBBA2:AF109682 GBHTG2:AC006759 GBHTG2:AC006759 GBBA1:MTY2OB11 GBBA1:XANXANAB GBGSSIO:AQ194038 GBBA1:MTY2OBiI GB-GSS3:BlG0037 GB-GSS3:B09549 GB-BA1 :MTCY71 GBHTG5:AC007547 GB-HTG5:AC007547 GBBA2:AF072709

GB-BAI:CGLYSEG

GB-PR4:AC005906 GB-BAI :CGPTMACKA GBBA1:D90861 GB-PAT:E02087 990 103725 103725 36330 3410 697 36330 974 1097 42729 262181 262181 8366 2374 185952 3657 14839 1200 280 3.49 AF109682 AC006759 AC006759 Z95121 M83231 AQ194038 Z95 121 B10037 B09549 Z92771 AC007547 AC007547 Aquaspirillum arcticum malale dehydrogenase (MDH) gene, complete cds.

Caenorhabditis elegans clone Y40G1I2, SEQUENCING IN PROGRESS-, 8 unordered pieces.

Caenorhabditis elegans clone Y40G12, SEQUENCING IN PROGRESS-, 8 unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome; segment 139/162.

Xanthomon *as campestris phosphoglucomnutase and phosphomannomnutase (xanA) and phosphomnannose isomnerase and GDP-mannose pyrophosphorylase (xanB) genes, complete cds.

RPCI1 1-47D24.TJ RPCI-1 1 Homo sapiens genomlc clone RPCI-1I 1 -47D24.

genomnic survey sequence.

Mycobacteriumn tuberculosis H-37Rv complete genome; segment 139/162.

T27A1 9-T7 TAMU Arabidopsis thaliana genomlc clone T27A1 9. genomic survey sequence.

T21A19-T7.1 TAMU Arabidopsis thaliana genomic clone T21A19. genomic survey sequence.

Mycobacteriumn tuberculosis H37Rv complete genome; segment 141/182.

Homo sapiens clone RP1 1-252018, WORKING DRAFT SEQUENCE. 121 unordered pieces.

Homo sapiens clone RP1 1-252018, WORKING DRAFT SEQUENCE, 121 unordered pieces.

Aquaspirillum arcticum Caenorhabdltis elegans Caenorhabdilis elegans Mycobacterium tuberculosis Xanlhomonas campestris Homo sapiens Mycobacterium tuberculosis Arabidopsis thaliana Arabidopsis thaliana Mycobacterium tuberculosis Homo sapiens Homo sapiens Streplomyces lividans Corynobaclerium glulamicum Homo sapiens Corynebacterium glulamicum Escherichla col Escherichia coli Helicobacter pyloni Homo sapiens 34,821 58,487 37,963 37,963 38,011 47,726 36,599 36,940 35,284 38,324 39,778 32,658 38,395 55,221 100,000 36,756 100.000 53.041 54,461 39,286 39,412 1 6-Sep-99 19-OCT-1999 25-Feb-99 25-Feb-99 17-Ju 1-98 26-Apr-93 20-Apr-99 17-Jun-98 14-MAY-i1997 14-MAY-i 997 10-Feb-99 16-Nov-99 16-Nov-99 8-Jul-98 24-Feb-97 30-Jan-99 23-MAR-i1999 29-MAY-i1997 29-Sep-97 9-Apr-97 3-Jun-99 AF072709 Streptomyces lividans amplifiable element AUD4: putative transcriptional regulator, putative ferredoxin, putative cytochrome P450 oxIdoreductase, and putative oxidoreductase genes, complete cds; and unknown genes.

X96471 C.glutamicum lysE and lysG genes.

AC005906 Homo sapiens 12p13.

3 BAC RPCI1 1-429A20 (Roswell Park Cancer Institute Human BAC Library) complete sequence.

X89084 C.giutamicum pta gene and ackA gene.

D90861 Ecoli genomic DNA, Kohara clone #405(52.0-52.3 min.).

E02087 DNA encoding acetate kinase protein form Escherichia coli.

U60627 Helicobacter pylon feoB-like DNA sequence, genomic survey sequence.

A1701691 we8lcO4.xi SoaresNFL_-T_-GBC -S I Homo sapiens cDNA clone IMAGE:2347494 3 similar to gb:Li 9686_ra I MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);, mRNA sequence.

rxa01468 948 GB-GSS1:HPU60627 GBEST31 :Al701691 2007203039 29 Jun 2007 GB-EST1:AA480256 389 nxa01478 1959 GBBA1:SC151 GB-BAI:SCE36 GB-BA1 CGU43535 nxa01482 1998 GBBA1:SC6G4 GBBA1:U00020 GBtBA1:MTCY77 40745 12581 2531 41055 36947 22255 Table 4 (continued) AA480256 ne311f04.sl NCICGAPCo3 Homo sapiens cONA clone IMAGE:898975 3' similar to gb:LI9686jrna1 MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);, mRNA sequence.

AL109848 Streptomyces coelicolor cosmld 151.

AL049763 Streptomyces coeticolor cosmId E36.

U43535 Cory nebacterlum glutamicum multidrug resistance protein (cmr) gene, complete cds.

AL031317 Streptomyces coeticolor cosmid 6G4.

U00020 Mycobacterium leprae cosmid B229.

Z95389 Mycobacterium tuberculosis H37Rv complete genome: segment 146/1 62.

H-omo sapiens Streptomyces coelicolor A3(2) Slreptomyces coellcolor Corynebacterumn glutamicum Streplomyces coelicolor Mycobacterlum leprae Mycobacterium tuberculosis 54,141 38,126 41,852 62,149 38,303 38,179 39,574 14*Aug-97 16-Aug-99 05-MAY-1999 9-Apr-97 20-Aug-98 01-MAR-1994 18-Jun-98 rxa01 534 rxa0iS35 1530 rxa0155O 1635 GBBA1:MLC81222 GB-BAI:MTV0l7 GBBAI:PAU72494 GBBA1:D90907 GBJIN2:AF0731 77 GB3IN2:AF0731 79 34714 67200 4368 132419 9534 3159 AL049491 AL021 897 U72494 D90907 AF0731 77 AF0731 79 Mycobacterium leprae cosmid B 1222. Mycobacterium leprae Mycobacterium tuberculosis H37Rv complete genome; segment 48/1 62. Mycobacterium tuberculosis Pseudomonas aeruglnosa fumarase (fumC) and Mn superoxide dismulase Pseudomonas aeruginosa (sodA) genes, comptete eds.

Synechocystis sp. PCC6803 complete genome. 9/27, 1056467-1158885. Synechocystls sp.

Drosophila melanogaster glycogen phosphorylase (GlyP) gene, complete cds, Drosophila melanogaster Drosophila melanogaster glycogen phosphorylase (Glpi) mRNA, complete cds. Drosophila melanogasler 66,208 38,553 52,690 56.487 55. 100 56,708 27-Aug-99 24-Jun-99 23-OCT-1996 7-Feb-99 1-Jul-99 27-Apr-99 rxaOl 562 rxa01569 1482 GBBAI:D78182 GBBA2:AF0791 39 GBBA2:AF087022 rxaOlS7O 978 GB-BAI:MTCY63 GB-BA2:AF097519 7836 4342 1470 38900 4594 D78 182 AF079 139 AF087022 Z96800 AF09751 9 Streptococcus mutans DNA for dTDP-rhamnose synthesis pathway, complete cds.

Streptomyces venezuelae pikCD operon, complete sequence.

Streptomyces venezuelae cytochrome P450 monooxygenase (picK) gene, complete cds.

Mycobacterium tuberculosis H-37Rv complete genome; segment 16/162.

Klebsiella pneumonlae dTDP-D-glucose 4,6 dlehydratase (rmlB). glucose-i phosphate thymldylyl transferase (rmtA), dTDP-4-keto-L-rhamnose reduclase (rmlD). dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase (rmlIC). and rhamnosyl transferase (wbbL) genes, complete cds.

Streptococcus mutans Streplomyces venezuelae Streptomyces venezuelae Mycobacteriumn tuberculosis Klebsiella pneumoniae 44.050 38,587 38,621 59,035 59,714 5-Feb-99 28-OCT-i1998 1 5-OCT-1998 17-Jun-98 4-Nov-98 2007203039 29 Jun 2007 GBBA2:NGOCPSPS rxa01571 723 GB-BA1:AB01 1413 GBBA1:ABOI 1413 rxa01572 615 GB-BAI:A8011413 GB-BA:ABO1 1413 Table 4 (continued) 8905 L091 89 Neisseria meningilidis dTOP-D-glucose 4.6-ehydratase (ribB), glucose-i- Neisseria meningitidis phosphate thymidyl transterase (rnbA) and rfbC genes. compiete cds and UjPDglucose.4-epimerase (galE) pseudogene.

12070 ASO011413 Streptomyces griseus genes for Orf2, OrfG. Orf4, Orf5, AfsA, Orf8, partial and Streptomyces griseus complete cds.

12070 ABOl 1413 Streplomyces griseus genes for Orf2, Qrf3, Orf4, 0(f5, AfsA, OrfB, partial and Streptomyces griseus complete cds.

12070 AB01 1413 Streptomyces griseus genes for Orf2, Orf3, Orf4, Orf5, AfsA, OrfB. partial and Streptomyces griseus complete cds.

12070 ABO1 1413 Streplomyces gniseus genes for Orf2. Orf3, Orf4. Orf5, AfsA, Orft. partial and Streplomyces griseus complete cds.

4783 U72240 Choristoneura fumniferana nuclear polyhedrosis virus ETM protein homolog, 79 Choristoneura fumiferana kDa protein homnolog, 15 kDa protein hamolog and GTA protein homolog nucleopolyhedrovirus genes, complete cds.

408 AQ213248 HS_3249_81_A02 MR CITApproved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomic clone Plate=3249 Coi=3 Row=B. genomic survey sequence.

58,384 30-Jul-98 57,500 35,655 57,843 38,119 37,115 7-Aug-98 7-Aug-98 7-Aug-98 7-Aug-98 29-Jan-99 rxaOl6OG 2799 GBVI:CFU72240 GBGSS1O:AQ213248 GBGSS8:A0070145 285 34.559 18Sp9 40,351 5-Aug-98 rxa01626 468 GB-PR4:AF152510 GBPR4:AF152323 GBPR4:AF152509 ra0632 1128 GBHTG4:AC006590 2490 4605 2712 127171 GBHTG4:AC006590 127171 A0070145 HS_3027_Bi_HO2_MR CIT Approved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomic clone Plate=3027 Col=3 Row=P, genomic survey sequence.

AF152510 Homo sapiens protocadherin gamma A3 short form protein (PCDH-gamma-A3) Homo sapiens variable region sequence, complete cds.

AF152323 Homo sapiens protocadherin gamnma A3 (PCDH-gamma-A3) mRNA, complete Homo sapiens cds.

AF152509 Homo sapiens PCDH-gamma-A3 gene, aberrantly spliced, mRNA sequence. Homo sapiens AC006590 Drosophila melanogaster chromosome 2 clone BACR13NO2 (0543) RPCI-98 Drosophila mel 13.N.2 map 36E-36E strain y; cn bw sp, -SEQUENCING IN PROGRESS-, 101 unordered pieces.

AC006590 Drosophila melanogaster chromosome 2 clone BACR13NO2 (D543) RPCI-98 Drosophila mel 13.N.2 map 36E-36E strain y; cn bw sp, -SEQUENCING IN PROGRESS-, 101 unordered pieces.

B99182 CIT-HSP-2280113.TR CIT-HSP Homo sapiens genomic clone 2280113, Homo sapiens genomic survey sequence.

Z991 12 Bacillus subtilis complete genome (section 9 of 2 from 1598421 to 1807200. Bacillus subtilis Z991 12 Bacillus subtilis complete genome (section 9 of 21): from 1598421 to 1807200. Bacillus subilis ACD06247 Drosophila melanogaster chromosome 2 clone BACR48110 (0D505) RPCI-98 Drosophila met 48.1.10 map 49E6-49F8 strain y; cn bw sp, SEQUENCING IN PROGRESS unordered pieces.

anogaster anogaster 33,812 34,298 34.298 34,298 33,812 14-Jul-99 22-Jul-99 14-Jul-99 19-OCT- 1999 19-OCT-i1999 26-Jun-98 26-Nov-97 26-Nov-97 2-Aug-99 rxa01633 1206 GBG558:B99182 GBBA1:BSUB0009 GBBAI:BSUBOOO9 GB-HTG2:AC006247 415 208780 208780 174368 anogaster 36.111 36,591 34,941 37.037 2007203039 29 Jun 2007 Table 4 (continued) rxaOlI695 lbZ3 GB b3A1:CUGA224946 24U8 AJ22494b Coryneacterium glutamicum DNA for L-Malate:qulnone oxidoreductase. Corynebacterium glutamicum GBBA1:MTCY24A1 GBIN1:DMU15974 rxaOl7O2 1155 GBBA1:CGFDA GBBA1:MTY13E1O GBBA1:MLCB4 rxa01743 901 GB_1N2:CELC27H5 GBEST24:Al167112 GB-GSS9:AQ1 02635 rxa01744 1662 GBBA1:MTCYOIB2 GBGSS I:AF009226 GBBAI:5CD78 rxa01745 836 GBBAI:MTCYI9O GBBA1:MLCB22 GBBA2:AE000175 rxa01758 1140 GBPR3:HS57G9 GBPL2:YSCH9666 GBPL2:YSC-9986 rxaOl8l4 1765 GB-BA1:ABCCELB GBBA1:MTCY22D7 GBBA1:MTCY22D7 rxa0185i 1809 GBGSS9:AO142579 20270 2994 3371 35019 36310 35840 579 3.47 35938 665 36224 34150 40281 15067 113872 39057 41664 2058 31859 31859 529 108924 637 Z95207 U 15974 X17313 Z95324 AL-02351 4 U 14635 A1167 112 AQ102635 Z95554 AF009226 AL034355 Z70283 Z98741 AE0001 75 Z951 16 U 10397 U00027 L24077 Z83866 Z83866 AQ 142579 AC005889 AGO08814 Mycobactefiumn tuberculosis H37Rv complete genome; segment 124/162. Mycobacterium tuberculosis Drosophlla melanogaster kinesin-like protein (ktp68d) mRNA, complete cds. Drosophila metanogaster Corynebaclerlum glutamicum fda gene for fructose-bisphosphate aldolase (EC Corynebactenium 4.1.2.13). glutamicum' Mycobacterium tuberculosis H37Rv complete gonome; segment 18/1 62. Mycobacterium tuberculosis Mycobacterium Ieprae cosmid 84. Mycobacterium leprae Caenorhabditls elegans cosmld C271-5. Caenothabditis elegans xylem.est.878 Poplar xylem Lambda ZAPII library Populus balsamifera subsp. Populus balsamifera trichocarpa 6DNA mRNA sequence. subsp. trlchocarpa HS_7304881,j08_MF CIT Approved Human Genomic Sperm Library 0 Home Home sapiens sapiens genomic clone Plate=3048 001= 15 Row=L, genomic survey sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 72/1 62. Mycobacterium tuberculosis Mycobacterium tuberculosis cytochrome D oxldase subunit I (appC) gene. Mycobacterium partial sequence, genomlc survey sequence. tuberculosis Streptomyces coelicolor cosmid 078. Streptomyces coelicolor Mycobacterium tuberculosis H37Rv complete genome; segment 981162. Mycobacterium tuberculosis Mycobacterium leprae cosmld 822. Mycobacterium leprae Escherichla cell K-12 MG1655 section 65 of 400 of' the complete genome. Escherichla coli Human DNA sequence from BAG 57G9 on chromosome 22q 12.1 Contains Home sapiens ESTs, CA repeat, GSS.

100,000 38,626 36,783 99.913 38,786 38,238 35,334 39,222 40.653 36,650 63,438 53,088 62,081 61,364 52.323 39,209 11 -Aug-98 17-Jun-98 18-Jul-95 12-Sep-93 17-Jun-98 27-Aug-99 13-Jul-95 03-DEC-i1998 27-Aug-98 17-Jun-98 3 1-Jul-97 26-Nov-98 17-Jun-98 22-Aug-97 12-Nov-98 23-Nov-99 5-Sep-97 29-Aug-97 21 -Sep-94 17-Jun-98 17-Jun-98 24-Sep-98 30-OCT- 1998 7-Feb-99 Saccharomyces cerevisiae chromosome ViIl cosmid 9666. Saccharomyces cerevisiae 40,021 Saccharomyces cerevislae chromosome ViII cosmid 9986. Saccharomyces cerevisiae 34,375 Acetobacter xylinum phosplioglucomutase (ceIB) gene, complete cds. Acotobacter xylinus 62,173 Mycobacterium tuberculosis H37Rv complete genome; segment 133/162. Mycobacterium 39,749 tuberculosis Mycobacterium tuberculosis H37Rv complete genome: segment 133/1 62. Mycobacterlum 40,034 tuberculosis HS_2222_81_H03_MR CIT Approved Human Genomic Sperm Library D Home Home sapiens 38.068 sapiens genomic clone Plate=2222 Col=5 Row=P, genomic survey sequence.

Drosophila melanogaster, chromosome 2L, region 30A3- 30A6. P1 clones Drosophila melanogasler 36,557 DS06958 and DS03097, complete sequence.

Home sapiens genomic DNA, 21q region, clone: 8137878868, genomic survey Home sapiens 35,316 sequence.

GBJIN2:AC005889 GBGSSl-AG008814 2007203039 29 Jun 2007 rxa01859 1050 GBBA2:AF 183408 GBHTG5:AC0080311 GB-BA2:AF183408 ra01865 438 GBBA1:SERFDXA GBBAI:M1VOOS GBBA1:MSGY348 rxa01882 1113 GB-PR1:HUMADRA2C GBPR4:HSU72648 GB-GSS3:B42200 rxa01884 1913 GB-BA1:MTCY48 GB-BA1 :SCOOO1 206 GBBA1:090908 rxa01886 897 GBGSS9:AQI 16291 GBBA2:AE001721 GB-EST16:AA567090 rxa01887 1134 GB-HTG6:AC008147 GB-HTG6:AC008147 GB-BA2:ALW243431 53626 AF183408 158889 AC008031 63626 AF183408 3869 M61119 37840 AL010186 40058 AD000020 1491 J03853 4850 U72648 387 B42200 35377 Z74020 9184 AJO01206 122349 090908 572 AQl 16291 17632 AE001721 596 A567090 303147 AC008147 303147 AC008147 26953 AJ243431 Table 4 (continued) Microcystis aenjginosa DNA polymerase III beta subunit (dnaN) gene, partial cds; microcystin syntnetase gene cluster, complete sequence; Uma 1 (uma 1), Uma2 (uma2), Uma3 (uma3), Uma4 (uma4), and Uma5 (uma5) genes.

complete cds; and Uma6 (uma6) gene, partial cds.

Trypanosoma brucel chromosome 11 clone RPCI93-25N 14, -SEQUENCING IN PROGRESS 2 unordered pieces.

Microcystis aeruginosa DNA polymerase IlI beta subunit (dnaN) gene, partial cds: microcystin synthetase gene cluster, complete sequence; Umal (uma 1), Uma2 (uma2), Uma3 (uma3), Uma4 (uma4l), and Uma5 (uma5) genes, complete cds; and Uma6 (uma6) gene, partial cds.

Saccharopotyspora erylhraea ferredoxin (fdxA) gene, complete cds.

Mycobaclerlum tuberculosis H37Rv complete genome; segment 511162.

Mycobacterium tuberculosis sequence from clone y348.

Human kidney alpha-2-adrenerglc receptor mRNA. compiete cds.

H-omo sapiens alpha2-C4-adrenerglc receptor gene, complete cds.

HS-1055-B1-A03-MR.ab CIT Human Genomic Sperm Library C Homo sapiens genornic clone Plate=CT 777 Cot=5 Row-B, genomic survey sequence.

Mycobacteriumn tuberculosis H37Rv complete genome; segment 69/162.

Streplomyces coelicolor A3(2), glycogen metabolism cluster IL.

Synechocystis sp. PCC6803 complete genome, 10/27, 1188886-1311234.

RPCI1 1-49P6.TK.l RPCI-1 I Homo sapiens genomic clone RPCI-1 1-49P6.

genomic survey sequence.

Thermotoga maritima section 33 of 136 of the complete genome.

GM01044.5prime GM Drosophila melanogaster ovary BlueScript Drosophila melanogaster cDNA clone GM01 044 5prime, mRNA sequence.

Homo sapiens clone RP3-405J10, SEQUENCING IN PROGRESS ~.102 unordered pieces.

Homo sapiens clone RP3-405J110, -SEQUENCING IN PROGRESS ,102 unordered pieces.

AcInetobacter Iwoffii wzc, wzb, wza, weeA, weeB, wceC, wzx, wzy. weeD, weeE, weeF, weeG, wed-I, weel, weeJ, weeK. galU, ugd, pgi, galE, pgmn (partial) and mlp (partial) genes (emulsan blosynthetic gene cluster), strain RAG-i1.

Microcystis aeruginosa Trypanosoma brucei Microcystis aeruginosa Saccharopolyspora erylhraea Mycobacterium tuberculosis Mycobacterlum tuberculosis Homo sapiens Homo sapiens Homo sapiens Mycobacterium tuberculosis Streptomyces coelicolor Synechocystis sp.

Homo sapiens Thermotoga maritima Drosophila melanogaster Homo sapiens Homo sapiens Acinetobacter twoffli 36,36>4 35,334 36,529 59,862 61,949 59,908 36,899 36.899 34.805 37,892 40,413 47,792 43,231 39.306 42,807 36,417 37,667 39,640 03-OCT-1999 1 5-Nov-99 03-OCT-1999 13-MAR-1996 17-Jun-98 10-DEC-1996 27-Apr-93 23-Nov-98 18-OCT-i 997 1 7-Jun-98 29-MAR- 1999 7-Feb-99 20-Apr-99 2-Jun-99 28-Nov-98 03-DEC-1999 03-DEC-i1999 01-OCT-1999 2-Aug-99 rxa0lBB8 658 GBHTG2:AC008197 125235 AC008197 Drosophila rnelanogaster chromosome 3 clone BACR02112 (0753) RPCI-98 02.12 map 9413-94C strain y; cn bw sp, SEQUENCING IN PROGRESS-, 113 unordered pieces.

Drosophila melanogaster 32.969 2007203039 29 Jun 2007 GBHTG2:AC008197 GBEST36:A1881527 rxa01891 887 GBVI:H1V232971

GBPLI:AFCHSE

GB-PR3:AF064858 rxa01895 1051 GBBA1:CGL238250 GBBA2:AF038423 GBBA1:MTCY359 rxa01901 1383 GBBA1:MSGB38COS GBBA1:S0E63 GBPR3:AF0931 17 rxa01927 1503 GB-BA1:CGPAN GBBA1:ASXYLA GBHTG3:AC009500 rxa01952 1836 GBBA2:AE000739 GB-EST28:A51 9629 GBEST21:AA949396 rxa01989 630 GBBA1:BSPGIA GBBAI:BSUBO017 GBBA2:AF132127 rxa02026 720 GB-BA1:SXSCRBA GBBAi:BSUBO02O GB BA1 :BSGENR rxa02028 526 GBBA1:MTC1237 125235 AC008197 598 A1881527 621 AJ232971 6158 Y09542 193387 AF064858 1593 AJ238250 1376 AF038423 36021 Z83859 37114 L01095 37200 AL035640 147216 AF093117 2164 X96580 1905 X59466 176060 ACO00500 13335 AE000739 612 A1519629 767 AA949396 1822 X16639 217420 Z99120 8452 AF132127 3161 X67744 212150 Z99123 97015 X73124 27030 Z94752 Table 4 (continued) Drosophila melanogaster chromosome 3 clone BACRO2LI12 (D753) RPCI-98 Drosophila melanogaster 021L.12 map 9413-94C strain y; cn bw sp, -SEQUENCING IN PROGRESS -,113 unordered pieces.

6060700 0 9.yl 606 Ear tissue cONA library from Schmidt lab Zea mays cDNA, Zea mays mRNA sequence.

Human immunodeficiency virus type 1 subtype C net gene, patient MP83.

Afumigatus chsE gene.

Homo sapiens chromosome 21q22.3 BAG 28F9, complete sequence.

Corynebaclerium glulamlicumn ndh gene.

Mycobacterium smegmatis NADH dehydrogenase (ndh) gene. complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 84/1 62.

M. leprae genomic DNA sequence. cosmid 838 bfr gene, complete cds.

Streptomyces coelicolor cosmid E63.

Homo sapiens chromosome 7qtelo BAG E3, complete sequence.

C.glutamicum panB, panG xylB genes.

Arthrobacter Sp. N.R.R.L. 03728 xylA gene for D-xylose(D-glucose) Isomerase.

Homo sapiens clone NHO51 1A20. SEQUENCING IN PROGRESS 6 unordered pieces.

Aquifex aeollcus section 71 of 109 of the complete genome.

LD39282.5prlme LD Drosophila melanogaster embryo pOT2 Drosophila melanogaster cONA clone LD39282 5prime, mRNA sequence.

LD28277.5prlme LD Drosophila melanogasler embryo pOT2 Drosophila melanogaster cDNA clone LD28277 5prime, mRNA sequence.

Bacillus stearothermophilus pgiA gene for phosphoglucoisomerase isoenzyme A (EC 5.3.1.9).

Bacillus subtilis complete genome (section 17 of 21): from 3197001 to 3.414420.

Human immunodeficiency virus type 1 Aspergillus fumnigatus Homo sapiens Corynebacterlum glutamicum Mycobacterium smegmatis Mycobacterlum tuberculosis Mycobacterium leprae Streptomyces coelicolor Homo sapiens Gorynebacterium glutamicum Arthrobacter sp.

Htomo sapiens Aquifex aeolicus Drosophila melanogaster Drosophila melanogaster Bacillus stearothermophilus Bacillus subtilis 32,969 43,617 40,040 37,844 37,136 100,000 65,254 40.058 59.551 39,468 39,291 38,384 56,263 37,593 36,309 41.941 39.855 66,292 37.255 63,607 67,778 35.574 51,826 54,476 2-Aug-99 21-Jul-99 05-MAR-i1999 I1-Apr.97 2-Jun-98 24-Apr-99 05-MAY-i1998 17-Jun-98 6-Sep-94 17-MAR-I1999 02-OCT-i1998 1 1-MAY-1999 04-MAY-i1992 24-Aug-99 25-MAR-i1998 16-MAR-i1999 25-Nov-98 20-Apr-95 26-Nov-97 28-Sep-99 28-Nov-96 26-Nov-97 2-Nov-93 17-Jun-98 Streptococcus mutans sorbitol phosphoenolpyruvate:sugar phospholransferase Streptococcus mutans operon, complete sequence and unknown gene.

S.xylosus scr8 and sc-rR genes. Staphylococcus xylosus Bacillus subtilis complete genome (section 20 of 21): from 3798401 to Bacillus subtilis 4010550.

B.subtilis genomic region (325 to 333). Bacillus subtills Mycobacterium tuberculosis H37Rv complete genome; segment 46/162. Mycobacterium tuberculosis 2007203039 29 Jun 2007 rxa02054 1140 rxa02O56 2891 rxaO2O6l 1617 rxa02063 1350 rxaO21OO 2348 rxa02122 822 rxaO2l4O 1200 rxa02142 774 G RPi.2:SCE9537 GBGSSl 3:AQ501177 GBBAI:MLCB1222 GB-BA1 :MTY1 3E1 2 GBBA1:MTU43540 GBPAT:E14601 GBBA1:DB4i02 GB-BA1:MTV0O6 GBHTG7:AC005883 GBPL2:ATAC003033 GBYPL2:ATAC002334

GBBAI:SCGLGC

GBGSS4:AQ687350 GBE5T38:AW028530 GBBA1:MSGYI51 GB-BA1 :MTCY1 30 GBBA1:SC0001205 GB-BAI :090858 GBEST37:A1948595 GBHTG3:AC010387 66030 767 34714 43401 3453 4394 4394 22440 211682 84254 75050 1518 786 444 37036 32514 9589 13548 469 220665 U18778 AQ501177 AL049491 Z95390 U43540 E14601 084102 AL021 006 AC005883 AC003033 AC002334 X89733 A0687350 AW028530 A00000118 Z73902 AJO01 205 D90858 A1948595 AC010387 178813 L78814 AF093099 Z70283 Table 4 (continued) Saccharomyces cerevislae chromosome V cosmlds 9537, 9581. 9495, 9867, Saccharomyces cerevisiae 36,100 and lambda clone 5898.

V26G9 mTn-3xHA/lacZ Insertion Library Saccharomyces cerevisiae genomic Saccharomyces cerevisiae 32,039 genomic survey sequence.

Mycobacterium leprae cosmid B1222. Mycobacterium leprae Mycobacterium tuberculosis H-37Rv complete genome; segment 1471162. Mycobacterium tuberculosis Mycobacterium tuberculosis rfbA, mtamnose biosynthesis protein (rfbA). and Mycobacterium rmtC genes, complete cds. tuberculosis Brevibacterium laclofermentum gene for alpha-ketoglutaric acid Corynebacterium dehydrogenase. glutamicum Corynebacterlum gtutamicumn DNA for 2-oxoglutarate dehydrogenase. complete Corynebacterium cds. glulamicum Mycobacterium tuberculosis H37Rv complete genome; segment 54/11162. Mycobacterium tuberculosis Homo sapiens chromosome 17 clone RP1 1-958E1 1 map 17. Homo sapiens SEQUENCING IN PROGRESS 2 ordered pieces.

Arabidopsis thaliana chromosome 11 BAG T21 L14 genomic sequence, complete Arabidopsis thaliana sequence.

Arabldopsis I1haliana chromosome 11 BAC F25118 genomic sequence, complete Arabidlopsis thaliana sequence.

S.coelicolor DNA for glgC gene. Streptomyces coelicolor nbxbOO74Hl 1 r CUGI Rice BAC Library Oryza saliva gerlomic clone Oryza saliva nbxbOO74Hi1 r, genomic survey sequence.

wv27fIO.xi NCI CGAPKidi 1 Homo sapiens cDNA clone IMAGE:2530795 3' Homo sapiens similar to WP:TO3G1 1.6 CED4874 mRNA sequence.

Mycobacterium tuberculosis sequence from clone yi51. Mycobacterium tuberculosis Mycobacterium tuberculosis H-37Rv complete genome; segment 59/162. Mycobacterium tuberculosis Streptomyces coelicolor A3(2) glycogen metabolism clusterl. Streptomyces coelicolor Ecoli genomic DNA, Kohara clone #401(51.3-51.6 min.). Escherichla coli wqO7dlI2.xlI NCL-CGAP.J1d 12 Homo sapiens cDNA clone IMAGE:2470583 Homo sapiens mRNA sequence.

Homo sapiens chromosome 5 clone CITB-H1_2074D8, -SEQUENCING IN Homo sapiens PROGRESS 77 unordered pieces.

Mycobacterium leprae cosmid BI 551 DNA sequence. Mycobacterium leprae Mycobacterium leprae cosmid B 1554 DNA sequence. Mycobacterlum leprae Mus musculus transcription factor TBLYM (Tbiym) mRNA, complete cds. Mus muscutus Mycobacterium tuberculosis l-37Rv complete genome: segment 98/1 62. Mycobacterium tuberculosis 61,896 59,964 59,659 98,928 98.928 39.265 37,453 37,711 37,711 56,972 40.696 36,795 40,156 55,218 38,475 38,586 37,259 38,868 51,399 51,399 36,683 57,292 27-Aug-99 17-Jun-98 14-Aug-97 28-Jul-99 6-Feb-99 18-Jun-98 08-DEC-i 999 19-DEC-i1997 04-MAR-1998 12-Jul-99 1 -Jul-99 27-OCT.1999 10-DEC-i1996 17-Jun-98 29-MAR-i1999 29-MAY-i1997 6-Sep-99 15-Sep-99 15-Jun-96 15-Jun-96 01-OCT-I1999 17-Jun-98 1-Aug-97 29-Apr-99 GB-BAI:MSGB1551CS 36548 GB-BAI:MSGB1554CS 36548 GBRO:AF093099 2482 GBBAI:MTCYI9O 34150 2007203039 29 Jun 2007 Table 4 (continued) Streptomyces coelicolor cosmid 6G 10. GB-BAl :SC6GIO 36734 AL049497 GBBAI:AB016787 5550 ra02143 1011 GB-t3Al:MTCY19O 34150 GBBAI:MSGB1S51CS 36548 GBBAI:MSGB1554CS 365-48 rxa02144 1347 GBBA1:MTCY19O 34150 GBHTG3:ACOI 1500_0 300851 GB-HTG3:AC01 15000 300851 rxa02147 1140 GBEST28:Ai492095 GBEST1O:MA157467 GBEST1O:AA157467 rxa02149 1092 GBPR3:HSBK277P6 GBBA2:EMB065R075 GB-EST34:A1789323 rxa02175 1416 GBBAI:CGGLTG GBBA1:MTCY31 GBBA1:MLCB57 rxa02196 816 GBRO:RATDAPRP GB-GSS8:AQ012162

GBRO:RATDAPRP

nxa02209 1694 GBBAI:AB025424 GBBA2:AF002133 485 376 376 61698 360 574 3013 37630 38029 2819 763 2819 2995 15437 ABO 16787 Z70283 178813 L788 14 Z70283 AC01 1500 AC01 1500 A1492095 AA1 57467 AA1 57467 AL1 17347 AFI 118423 A1789323 X661 12 Z731 101 Z99494 M76426 A0012 162 M76426 AB025424 AF0021 33 Pseudomonas putida genes for cytochrome o ubiquinol oxidase A-E and 2 ORFs, complete cds.

Mycobacterium tuberculosis H37Rv complete genome: segment 98/162.

Mycobacterium Ieprae cosmid B1551 DNA sequence.

Mycobacterium Ieprae cosmid B1554 DNA sequence.

Mycobacterium tuberculosis H37Rv ,complete genome: segment 98/162.

Homo sapiens chromosome 19 clone CIT978SKB 60E1 1, SEQUENCING IN PROGRESS 246 unordered pieces.

Homo sapiens chromosome 19 clone CIT978SKB_60E1 1. SEQUENCING IN PROGRESS 246 unordered pieces.

tg0la0l.xl NCILCGAP..CLL1 Homo sapiens cONA clone IMAGE:2108040 3', mRNA sequence.

zoS0e0l.rl Stratagene endothelial cell 937223 Homo sapiens cONA clone IMAGE:590328 5% mRNA sequence.

zo50e0l .rl Stratagene endlothelial cell 937223 H-omo sapiens cONA clone IMAGE:590328 mRNA sequence.

Human DNA sequence from dlone 277P6 on chromosome 1q25.3-31.2, complete sequence.

Rhizobiumn etli mutant MB045 RosR-transcriptionally regulated sequence.

uk53g05.yl Sugano mouse kidney mkla Mus muscuius cONA clone IMAGE:1 972760 5' similar to WP:K1 1Hi12.8 CEI21 60;, mRNA sequence.

Cgiutamicumn gIt gene for citrate synthase and ORF.

Mycobacteriurm tuberculosis H37Rv complete genome; segment 41/162.

Mycobacterium ieprae cosmid 857.

Raltus norvegicus dipeptidyl amino peptid ase- related protein (dpp6) mRNA, complete cds.

127PB037070197 Cosmid library of chromosome If Rhodobacter sphaeroides genomic clone 127131037070197, genomic survey sequence.

Raltus norvegicus dipeptldyl aminopeptidase-related protein (dpp6) mRNA.

complete cds.

Corynebacterium glutamicum gene for aconitase. partial cds.

Mycobacterium avium strain GiRlO transcriptional reguiator (may81) gene, partial cds, aconitase (acn), invasin 1 (invi), invasln 2 (inv2), transcriptional regulator (moxR). ketoacyl-reductase (fabG), enoyl-reductase (InhA) and ferrochelalase (mav272) genes, complete cds.

Streplomyces coelicolor Pseudomonas pulida Mycobacteriumn tuberculosis Mycobacterium leprae Mycobacteriumn leprae Mycobacterium tuberculosis Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Rhizobium etli Mus musculus Corynebacterum giutamicum Mycobacteriurn tuberculosis Mycobacterium Ieprae Rattus norvegicus Rhodobacter sphaeroides Rattus norvegicus Corynebacterum giutamicum Mycobacterium avium 35,058 47,403 57,317 38,159 38,159 55.530 39,659 39.659 39,798 36.436 36,436 36,872 43,175 39,715 100.000 64,331 62,491 38,791 40.044 37,312 99,173 40.2 19 24-MAR.1999 5-Aug-99 17-Jun-98 15-Jun-96 1 5-Jun-96 1 8-Feb-00 18-Feb-00 30-MAR-1999 1 1-DEC-1996 1 1-DEC-1996 23-Nov-99 06-DEC-1999 2-Jul-99 17-Feb-95 17-Jun-98 10-Feb-99 31-MAY-1995 4-Jun-98 31 -MAY-1995 3-Apr-99 26-MAR-1998 2007203039 29 Jun 2007 (38_I:A1:MTV(UU7 rxa022i3 874 GBBA1:A8025424 GBBAI:MTVOO7 GBBA2:AF002 133 rxa02245 780 GB-BA2:RCU23145 GB-BA1:ECU82664 GBHTG2:AC007922 rxa02256 1125 GBBA1:CGGAPPGK GB-BA1:SCC54 GBBAI:MTCY493 rxa02257 1338 GB-BA1:CGGAPPGK GBBA1:MTCY493 GB.BA2:MAU82749 rxa02258 900 GB_BAl :CGGAPPGK GBBA1:CORPEPC GB-PAT:A09073 rxa02259 2895 GBBA1:CORPEPC GBPAT:A09073

GBBAI:CGPPC

32806 2995 32806 15437 AL021 184 AB025424 AL021 184 AF002 133 5960 U23145 139818 U82664 158858 AC007922 3804 30753 40790 3804 40790 2530 3804 4885 4885 4885 4885 3292 X59403 AL035591 Z95844 X59403 Z95844 U82749 X59403 M25819 A09073 M25819 A09073 X14234 Table 4 (continued) MywbaceiMu uerculosis H37,R-. comnplete genome; semmnt~n 641152. Mycnbacierium tuberculosis Corynebacterium glutamicum gene for aconitase. partial cds. Corynebacierium glutamicum Mycobacterium tuberculosis H37Rv complete genome; segment 641162. Mycobacterium tuberculosis Mycobacterium avlumn strain GIR10 ttanscriptional regulator (may81) gene. Mycobacterium avlum partial cds, aconitase (acn), invasin 1 (Invi). invasin 2 (inv2). transcriptional regulator (moxR). ketoacyl-reductase (fabG), enoyl-reductase (inhA) and ferrochelatase (mav272) genes, complete cds.

Rhodobacter capsulatus Calvin cycle carbon dioxide fixation operon: fructose- Rhodobacter capsutatus 1,6.-sedoheptulose.1.7-bispl1osphate aldolase (obbA) gene, partial cds. Form 11 ribulose-1 .5-bisphosphate carboxylasefoxygeflaSe (cbbM) gene.

complete cds, and Calvin cycle operon: pentose-5-phosphate-3-epimerase (cbbE), phosplioglycolate phosphatase (cbbZ), and cbbY genes, complete cds.

Escherichia coli minutes 9 to 11 genomic sequence. Escherichia coi HOMO sapiens chromosome 18 clone hRPK.178j_.,10 map '18, Homo sapiens SEQUENCING IN PROGRESS 11 unordered pieces.

C.glutamlcum gap, pgk and ipi genes for glyceraldehyde-3-phosphate, Corynebacterium phosphoglycerate kinase and trlosephosphate Isomerase. glutamicum Streptomyces coelicolor cosmid C54. Streptomyces coelicolor Mycobacterium tuberculosis H37Rv complete genome; segment 63/162. Mycobacterium tuberculosis C.glutamlcum gap, pgk and tpl genes for glyceraldehyde-3-phosphate, Corynebactenurri phosphoglycerate kinase and triosephosphate isomerase. glutamicum Mycobacterium tuberculosis H-37Rv complete genome; segment 631162. Mycobacterium tuberculosis Mycobacterium avium glyceraldehyde-3-phosphate dehydrogenase homolog Mycobacterium avium (gapdh) gene, complete cds; and phosphoglycerate kinase gene, partial cds.

C.glutamicumn gap, pgk and tpi genes for glyceraldehyde-3-phosphate, Corynebacerium phosphoglycerate kinase and Iriosephosphate isomerase. glutamicum C.giutamicum phosphoenolpyruvate carboxylase gene, complete cds. Corynebacterium giutamicum C.gluiamicum ppg gene for phosphoenol pyruvate carboxylase. Corynebacterium giutamicum C.glulamlcum phosphoenolpyruvate carboxylase gene, complete cds. Corynebacterium glutamicumn C.glutamicum ppg gene for phosphoenol pyruvate carboxylase. Corynebaclerium glutamlcum Corynebacterium glutamicum phosphoenoipyruvate carboxylase gene (EC Corynebacterium 4.1.1.31). giulamicum 38.253 99,096 34,937 36,885 46,701 39,119 33.118 99.289 36,951 64,196 98,873 61,.273 6 1.772 99,667 100,000 100,000 100.000 100,000 99,827 117-Jun-98 3-Apr-99 1 7-Jun-98 26-MAR-I1998 28-OCT-I1997 11-Jan-97 26-Jun-99 05-OCT-i1992 11-Jun-99 19-Jun-98 05-OCT-1 992 19-Jun-98 6-Jan-98 05-OCT-1992 15-DEC-1995 25-Aug-93 I15-DEC-1995 25-Aug-93 12-Sep-93 2007203039 29 Jun 2007 Table 4,(continued) 243145 AL050317 Human DNA sequence from clone RP1-94E24 on chromosome 20q12.

nxa02288 969 GBPR3:HSDJ94E24 GBHTG3:AC010091 GBHTG3:ACO1009I rxa02292 798 GBBA2:AF125164 GBGSS5:AQ744695 GBESTI4:AA381925 rxa02322 511 GB-BA1:MTCY22G8 GBBAI:MTCY22G8 nxa02326 939 GB-BAI:CGPYC GBBA2:AF03854 GBBA1:MTCY349 rxa02327 1083 GBBA1:CGPYC GBBA2:AF038548 GBBAI:MTCY349 mx02328 1719 GBBAI:CGPYC GBBA2:AF038548 GSBPL2:AF097728 rxa02332 1266 GBBAI:MSGLTA GB-BA2ABU85944 GB BA2:AEOOOI 75 =x02333 1038 GBBA1:MSGLTA 159526 159526 26443 827 309 22550 22550 3728 3637 43523 3728 3637 43523 3728 3637 3916 1776 1334 15067 1776 ACO 10091 AC010091 AF1 25164 AQ744695 AA38 1925 Z95585 Z95585 Y09548 AF03854 Z8301 8 Y09548 AF038548 Z83018 Y09548 AF038548 AF097728 X60513 U85944 AE000175 X60513 complete sequence.

Homo sapiens clone NH0295A0i, SEQUENCING IN PROGRESS ,4 unordered pieces.

Homo sapiens clone NH0295A01, SEQUENCING IN PROGRESS ,4 unordered pieces.

Bacteroides fraglis 638R polysaccharide B (PS B32) biosynthesis locus, complete sequence; and unknown genes.

HS_5505_A2_C06_5P6 RPCI-11 Human Male BAC Library Homo sapiens genomlo clone Plate=1 081 Col= 12 Row=E, genomic survey sequence.

EST95058 Activated T-cells I Homo sapiens cONA 5'end, rnRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/1 62.

Corynebactedrlu glutamicum pyc gene.

Corynebacterium glulamicum pyruvate carboxylase (pyc) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 131/162.

Corynebacterium glutamlcum pyc gene.

Coryneb acterium glutamicum pyruvale carboxylase (pyc) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome: segment 131/1 62.

Corynebacterium glutamicum pyc gene.

Corynebacterium glutamicum pyruvate carboxylase (pyc) gene, complete cds.

Aspergillus terreus pyruvate carboxytase (Pyc) mRNA, complete cds.

M.smegmalls glIA gene for citrate synthase.

Antarctic bacterium DS2-3R citrate synthase (clsy) gene, complete cds.

Escherlchia coli K-12 MG 1655 section 65 of 400 of the complete genome.

Msmegmatls gILA gene for citrate synthase.

Homo sapiens Chromosome 16 BAC clone CIT987-SKA-1 13A6 -complete Homo sapiens Homo sapiens Homo sapiens Bacleroides fragilis Homo sapiens Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis Corynebacterium glutamicum Corynebacterlum glutamicum Mycobacterium tuberculosis Corynebacterium glutamicum Corynebacterlumn glutamicum Mycobacterium tuberculosis Corynebacterium glutanicum Corynebacterium glutamlcum 36,039 35,331 35,331 39,747 39,185 35,922 57,677 37,14 3 100,000 100.000 37,363 99,259 99,259 41,317 100,000 100,000 03-DEC.1999 11 .Sep-99 11 -Sep-99 01-DEC-1999 16-Jul-99 21 -Apr-97 17-Jun-98 17-Jun-98 08-MAY-1998 24-DEC-1997 17-Jun-98 08-MAY-1998 24-DEC-i1997 17-Jun-98 08-MAY-I1998 24-DEC- 1997 29-OCT.1998 20-Sep-91 23-Sep-97 12-Nov-98 20-Sep-91 23-Nov-99 Aspergitlus terreus 52,248 Mycobacterium smegmatis 58,460 Antarctic bacterium 052- 57,154 3R Escherichia coil 38,164 Mycobacterium smegmatis 56,929 Hlomo sapiens 33,070 GBPR4:HUAC002299 171681 AC002299 genomic sequence, complete sequence.

2007203039 29 Jun 2007 GB-TG2:AC007889 rxa02399 1467 G8_BA1:CGACEA GB_BA1:CORACEA GBPAT:l113693 rxa02404 2340 GB-BAI:GGACEB

GB_BAI:COPACEB

GBBAl:PFFC2 rxa02414 870 GBPR4:AC007102 GB-HTG3:AC011214 GBHTG3:AC011214 rxa02435 681 GBBA2:AF101055

GB_OM:RABPKA

GBOM:RABPLASISM

rxa02440 963 GBEST14:AA417723 GBEST1l:AA215428 GBBA1:MTCY77 rxa02453 876 GBEST14:AA426336 GBBAi:STMMACC8 GBPR3:ACD04500 rxa02474 897 GB-BAI:AB009078 GB-OM:BTU71 200 GB_EST2:F12685 rxaO2480 1779 GBBAI:MTV012 127840 AC007889 Table 4 (continued) Drosophila melanogaster chromosome 3 clone BACR48E12 (13695) RPCI-98 Drosophila melanogaster 34.897 48.E.12 map 87A-87B strain y: cn bw sp, SEQUENCING IN PROGRESS-.

86 unordered pieces.

2427 X75504 C.giutamicum aceA gene and thiX genes (partial). Corynebacteflum glutamicum 1905 128760 Corynebacterium glutamicum isocitrate Iyase (aceA) gene. Corynebacterium giutamicum 2135 113693 Sequence 3 from patent US 5439822. Unknown.

3024 X78491 COglulamicum (ATCC 13032) aceB gene. Corynebactefium glutamicum 2725 L27123 Corynebacterlum glutamicum malate synthase (aceB) gene, complete cds. Corynebacterium glutamicum 5588 Y11998 P~fluorescens FC2.i, FC2.2, FC2.3c, FC2.4 and FC2.5c open reading frames. Pseudomonas fluorescens 176258 AC007102 Homo sapiens chromosome 4 clone C0162PI6 map 4p16, complete sequence. Homo sapiens 183414 AC01 1214 Homo sapiens clone LOW-PASS SEQUENCE SAMPLING. Homo sapiens 183414 AC01 1214 Homo sapiens clone 5_C_3, LOW-PASS SEQUENCE SAMPLING. Homo sapiens 7457 AF101055 Clostridium acetobutylicum-atp operon, complete sequence. Clostridium acetobutylicum 4441 J03247 Rabbit phosphorylase kinase (alpha subunit) mRNA, complete cds. Oryctolagus cuniculus 4458 M64656 Oryctolagus cuniculus phosphorylase kinase alpha subunit mRNA. complete Oryctolagus cuniculus cds.

374 AA417723 zv01b12.s1 NCI_COAP_GCBI Homo sapiens cONA clone IMAGE:746207 3' Homo sapiens similar to contains Alu repetitive etement;contains element Ll repetitive element;, mRNA sequence.

303 AA2 15428 zr95a07.s1 NOI_CGAP_GCBi Homo sapiens clDNA clone IMAGE:683412 3' H-omo sapiens similar to contains Alu repetitive element;, mRNA sequence.

22255 Z95389 Mycobacterium tuberculosis H37Rv complete genome, segment 1461162. Mycobacterlum tuberculosis 375 AA426336 zv53g02.sl Soares-testisNHT H-omo sapiens cDNA clone IMAGE:757394 3, Homo sapiens mRNA sequence.

1353 M55426 S.fradlae aminoglycoside acetyltransferase (aacC8) gene, complete cds. Streplomyces fradlae 77538 AC004500 Homo sapiens chromosome 5. PI clone 107689 (LBNL H14). complete Homo sapiens sequence.

2686 AB009078 Brevibacterium saccharolyticum gene for L-2.3-butanediol dehydrogenase, Brevibacterium complete eds. saccharoltyicum 877 U71 200 Bos taurus acetoln reductase mRNA, complete cds. Bos taurus 287 Ft12685 HSC30AO31 normalized infant brain cDNA Homo sapiens cONA clone c- Homo sapiens 3da03, mRNA sequence 70287 AL02 1287 Mycobacterium tuberculosis H37Rv complete genome: segment 132/162. Mycobacterium tuberculosis 100,000 100,000 99,795 99,914 99.786 63,539 35,069 36,885 36,885 39.605 36,061 36,000 38.770 39,93.4 38,889 38,043 37,097 33,256 96,990 51,659 41.509 36.737 2-Aug-99 9-Sep-94 10-Feb-95 26-Sep-95 13-Jan-95 8-Jun-95 11I-Jul-97 2-Jun-99 03-OCT-i 999 03-OCT.1999 03-MAR-I1999 27-Apr-93 22-Jun-98 16-OCT-i 997 13-Aug-97 1 8-Jun-98 16-OCT-1 997 05-MAY-1993 30-MAR-i1998 13-Feb-99 8-Oct-97 14-Mar-95 23-Jun-99 2007203039 29 Jun 2007 GBBA1:AP000060 36734 AL049497 347800 AP000060 rxa024B5 rxa02492 840 GBBAI:STMPGM GB-BA 1:MTCY2OG9 GBBA1:U00018 rxa02528 1098 GB-PR2:HS161N10 GB-HTG2:AC008235 GBHTG2:AC008235 rxa02539 1641 GBBA2:RSU17129 GBBAI;MTVO38 GB-BA2:AF068264 921 37218 42991 56075 136017 M83661 Z77 162 UDOO18 AL008707 AC008235 136017 AC008235 17425 16094 3152 rxa02551 483 GB-BA1:BACHYPTP 17057 GBBAl :BACH-UTWAP28954 GBBA1:BSGBGLUC 4290 rxa02556 1281 GBHTG3:AC008128 335761 GBHTG3:AC008128 335761 GB PL2:AC005292 99053 rxa02560 990 GBIN1:CEFO7A1 1 35692 GBE5T32:A1731605 566 GBINI:CEFO7A1 1 35692 U17129 AL021 933 AF068264 D29985 031856 Z34526 AC008 128 AC008 128 AC005292 Z66511 A173 1605 Z66511 Table 4 (continued) Streptomyces coelicolor cosmid 6G1.

Aeropyrumn pernix genomic DNA, section V/7.

Streptomyces coelicolor phosphoglycerate mutase (PGM) gene, complete cds.

Mycobacteriumn tuberculosis H37Rv complete genome; segment 25/162.

Mycobacterium leprae cosmid B21 68.

Human DNA sequence from PAC 161 N1 0 on chromosome Xq25. Contains

EST.

Drosophila reelanogaster chromosome 3 clone BACR1 5B19 (D995) RPCI-98 15.13.19 map 9417-95A strain y; cn bw sp. SEQUENCING IN PROGRESS 125 unordered pieces.

Drosophila melanogasler chromosome 3 clone BACRI 5819 (0995) RPCI-98 15.B.19 map 94F-95A strain cn bw sp, -SEQUENCING IN PROGRESS-, 125 unordered pieces.

Rhodococcus erythropolis ThcA (thcA) gene, complete cds; and unknown genes.

Mycobacterium tuberculosis H37Rv complete genome; segment 24/162.

Pseudomonas aeruginosa quinoprotein ethanol dehydrogenase (exaA)gene.

partial cds; cytochrome c550 precursor (exaB), NAD+ dependent acetaldehyde dehydrogenase (exaC), and pyrroloqulnoline qulnone synthesis A (pqqA) genes, complete cds; and pyrroloquinoline quinone synthesis B (pqqB) gene, partial cds.

Bacillus sublilis wapA and adf genes for wail-associated protein and hypothetical proteins.

Bacillus subtilis genome containing the hut and wapA loci.

B.subtliis (Marburg 168) genes for beta-glucoside permease and betaglucosidlase.

Homo sapiens. SEQUENCING IN PROGRESS ,106 unordered pieces.

Homo sapiens, SEQUENCING IN PROGRESS ',106 unordered pieces.

Genomic sequence for Arabidopsis thaliana BAC F261724, complete sequence.

Caenorhabdiis elegans cosmId FO7A1 1, complete sequence.

BNLGHi1 0201 Six-day Cotton fiber Gossyplumn hirsutumn cDNA 5' similar to (AC0O.4684) hypothetical protein [Arabidopsis thaliana), mRNA sequence.

Caenorhabdltls elegans cosmid FO7A1 1, complete sequence, Streptomyces coelicotor Aeropyrumn pernix Streptomnyces coelicolor Mlycobacterium ub:erculosis M1ycobacterium Ieprae Hlomo sapiens Drosophila melanogaster 35,511 48,014 65,672 6 1.436 37,893 37.051 36,822 Rhodococcus erythropolis Mvycobactedrn uberculosis Pseudomonas aeruginosa Bacillus subtilis Bacillus subtills Bacillus subtilis Homo sapiens Homo sapiens Arabidopsis thaliana Caenorhabdilis elegans Gossypium hirsutumn Caenorhabditis elegans 66,117 65,174 65,448 53,602 53,602 53,602 34,022 34,022 33,858 36,420 38,095 33.707 Drosophila melanogaster 36.822 24-MAR-1999 22-Jun .99 26-Apr-93 17-Jun-98 01 -MAR-i1994 23-Nov-99 2-Aug-99 2-Aug-99 16-Jul-99 17-Jun-98 18-MAR-1999 7-Feb-99 7-Feb-99 3-Jul-95 22-Aug-99 22-Aug-99 16-Apr-99 2-Sep-99 11-Jun-99 2-Sep-99 2007203039 29 Jun 2007 rxa02572 668 GBBA1:MTCY63 GBBA1:MTCY63 GBHTG1:HS24H01 rxa02596 1326 GBBA1:M1V026 GB_8A2:AF026540 GBBA2:MTU96128 rxa02611 1775 GBBA1:MTCY130 GBBA1:MSGY151 GBBA1:U00014 rxa02612 2316 GBBA1:MTCYI30 GBBA1:MSGY15I GBBA1:STMGLGEN rxa02621 942 GB-BA1:CGL133719 GBINI:CEM1O6 GBEST29:A1547662 rxa02640 1650 GBBA1:M1V025 GBBAI:PAU49666 GBBAI1A015974 rxa02654 1008 GBEST6:N65787 GB-PL2:Tl7H3 GBRO:MMU58105 rxa02666 891 GBPR3:AC004643 38900 38900 46989 23740 1778 1200 32514 37036 36470 32514 37036 2557 1839 39973 377 121125 4495 1641 512 65839 88871 43411 Z96800 Z96B00 AL12 1632 AL022076 AF026540 U96 128 Z73902 AD000018 U00014 Z73902 AD000018 L1 1647 AJ1337-19 Z46935 A1547662 AL022 121 U 49666 ABO1 5974 N65787 AC005916 U58 105 AC004643 Table 4 (continued) Mycobacterium tuberculosis H-31Rv complete genome; segment 16i 162. lMycobaclarlum, tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 16)'162. Mycobacterium tuberculosis Homo sapiens chromosome 21 clone LLNLc1 16H0124 map 21q21, Homo sapiens SEQUENCING IN PROGRESS In unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome; segment 157/162. Mycobacterium tuberculosis Mycobacterium tuberculosis UDP-galactopyranose mutase (glf) gene, complete Mycobacterium cds. tuberculosis Mycobacterlum tuberculosis UOP-galactopyranose mutase (gif) gene, complete Mycobacterium cds. tuberculosis Mycobacterium tuberculosis H-37Rv complete genome; segment 59/162. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone yl151. Mycobacterlum tuberculosis Mycobacterium leprae cosmid B1549. Mycobacterlum leprae Mycobacterium tuberculosis H37Rv complete genome; segment 59/162. Mycobacterium tuberculosis Mycobacterlum tuberculosis sequence from clone y151. Mycobacternum tuberculosis Streptomyces aureotaciens glycogen branching enzyme (glyB) gene, complete Streptomyces cds. aureofaciens Corynebacterium glulamnicumn yjcc gene, amtR gene and citE gene, partial. Corynebacterium glutamicum Caenorhabditis elegans cosmid M106, complete sequence. Caenorhabdilis elegans UI-R-C3-sz-h-03-0-U'lsi UI-R-C3 Rattus norvegicus clDNA clone UI-R-C3-sz-h- Rattus norvegicus 03-0-Ul mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome: segment 1551162. Mycobacterium tuberculosis Pseudomonas aeruginosa (orfX). glycerol dfiffuslon facilitator (glpF). glycerol Pseudomonas aeruginosa klnase (glpK), and Gip repressor (glpR) genes, complete cds, and (orfK) gene.

partial cds.

Pseudomonas tolaasil gIpK gene for glycerol kinase, complete cds. Pseudomonas tolaasii 20827 Lambda-PRL-2 Arabidopsis thaliana cDNA clone 232137T7. mRNA Arabidopsls thallana sequence.

Arabidopsis thaliana chromosome 1 BAG T171-3 sequence, complete Arabidopsis thaliana sequence.

Mus musculus Bilk locus, alpha-D-galactosidase A (Ags), ribosomal protein Mus musculus (1-44L). and Bruton's tyrosine kinase (Btk) genes, complete cds.

Homo sapiens chromosome 16, cosmid clone 363E3 (LANL). complete Homo sapiens sequence.

61,677 37,170 19,820 36,957 67,627 70,417 38.532 60.575 57,4866 38,018 58,510 57,193 36,858 37,608 50,667 39,187 59,273 58,339 39,637 33,735 35,431 38.851 17-jun-9OS 117-Jun.98 29-Sep-99 24-Jun-99 30-OCT-I1998 25-MAR-i1998 17-Jun-98 10-DEC-i1996 29-Sep-94 17-Jun-98 10-DEC-1996 25-MAY-I 995 12-Aug-99 2-Sep-99 3-Jul-99 24-Jun-99 18-MAY-I1997 28-Aug-99 5-Jan-98 5-Aug-99 13-Feb-97 01-MAY-i1998 2007203039 29 Jun 2007 GB-PR3:AC004643 43,411 AC004643 GBBA2:AF049897 9196 AF049897 rxa02675 1980 GB-BA1:PDENQOURF 10425 102354 GB-BA1:MTCY339 42861 Z77163 GSBA1:MXADEVRS 2452 L19029 rxa02694 1065 GB-BA1:BACLDH 1147 M19394 GB-BA1:BACLDHL 1361 M14788 GB-PATA06664 1350 A06664 rxa02729 844 GBEST15:AA494626 121 AA494626 GB-EST15:AA494626 121 A~A494626 Table 4 (continued) H-omo sapiens chromosome 16, cosmid clone 363E3 (LANL). complete sequence.

Corynebacterium glutamicum N-acetylglutamnylphosphate reduclase (argC).C ornithine acetyltransferase (argJ). N-acetylglutamate kinase (argB). 9 acetylornithlne transaminaso (argD), ornithine cartbamoyltransferase (argFl.

arginine repressor (argR). arginlnosuccinate synthase (argG). and arginlnosuccinate lyase (argH) genes, complete cds.

Paracoccus denitrificans NADH dehydrogenase (URF4). (NO8). (NQO9).

(URF6). (NQO1l0). (NOOI (N0012). (NQ013). and (NQO14) genes, complete cds's; biotin (acetyl-CoA carboxyl] 119550 (birA) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome: segment 101/162.

Homo sapiens 'orynebacterium ilutamicum Paracoccus denitrificans Miycobacterium uberculosis Myxococcus xanthus Bacillus catlolylicus Bacillus stearothermophilus Bacillus stearothermophilus Danio rerio Danio rerio 41.599 01-MAY-1998 40,4 13 1-Jul-98 Myxococcus xanlhus devR and devS genes, complete cds's.

B.caldolyticus lactate dehydrogenase (LDH) gene, complete cds.

B.stearothermophilus Ict gene encoding 1-lactate dehydrogenase. complete cds.

B.stearotherrnophilus let gene.

faOgdO4.rl Zebrafish lCRFzfls Danio rerio cDNA clone I 1A22 5' similar to TR:GI 171163 GI171163 G/T-MISMATCH BINDING PROTEIN.:. mRNA sequence.

Ia09d04.rl Zebralish ICRFzfls Danio rerio cDNA clone 1I1A22 5 similar to TR:G1 171163 GI 171163 G/T-MISMATCH BINDING PROTEIN. mRNA sequence.

I

40,735 36,471 38,477 57,371 57,277 57,277 50,746 20-MAY- 1993 17-Jun-98 27-Jan-94 28-Apr-93 26-Apr-93 29-Jul-93 27-Jun-97 V1y rxa02730 1161 GBEST19:AA758660 233 GBESTIS:A.A494626 121 AA758660 ah67d06.sI Soares testisNHT Homo sapiens cDNA clone 1320683 rnRNA Homo sapiens sequence.

A~A494626 Wa9dO4.rl Zebrafish ICRFzIIs Danio rerio cONA clone I 1A22 5' similar to Danio rerio TR:G1 171163 G1 171163 GIT-MISMATCH BINDING PROTEIN. mRNA sequence.

AC006285 Homo sapiens, complete sequence. Homo sapiens El 3655 gDNA encoding glucose-6-phosphate dehydrogenase. Corynebacteri.

36,364 27-Jun-97 37,059 29-DEC-1998 42,149 27-Jun-97 37,655 15-Nov-99 99.580 24-Jun-98 38,363 1 9-Jun-98 GBPR4:AC00)6285 GB-PAT:E13655 150172 2260 m rxa02737 1665 GBBA1:MTCY493 40790 Z95844 Mycobaclerium tuberculosis H37Rv complete genome; segment 63/162.

GBBAI:SC5A7 rxa02738 1203 GBPAT:E13655 GBBA1:SCC22 GBBA1:SC5A7 rxa02739 2223 GB-BA1:AB023377 40337 AL031 107 Streptomyces coelicolor cosmid 5A7.

2260 E 13655 gDNA encoding glucose-S-phosphate dehydrogenase.

Mycobacterium tuberculosis Streplomyces coelicolor Corynebacterium glutamicum Slreptomyces coelicolor Streplomyces coelicolor Corynebacteuium glutamicum 39.444 98,226 60,399 36,426 99.640 27-Jul-98 24-Jun-98 12-Jul-99 27-Jul.98 20-Feb-99 22115 40337 2572 AL096839 AL031 107 A8023377 Streptomyces coelicolor cosmid C22.

Streptomyces coelicolor cosmid 5A7.

Corynebacterium glulamicum tkt gene for lransketolase. complete cds.

2007203039 29 Jun 2007 GB BA1:MLCL536 GBBA1:U00013 rxa02740 1053 GBHTG2;A0006247 GBHTG2:AC006247 GBHTG3:AC007150 rxa02741 1089 GBHTG2:AC004951 GBHTG2:A0004951 GBINI:AB006546 rxa02743 1161 GBBAI:MLCL536 GBBAI:00013 GBHTG2:AC007401 rxa02797 1026 GBBA1:CGBETPGEN GBGSS9:AQ148714 GBBA1:BFU64514 rxa02803 680 GB-BA1:U00020 GBBA2:PSU85643 GB-BA1:SC6G4 nxa02821 363 GBHTG2:AC008105 GBHTG2:AC008105 36224 35881 174368 Z99 125 U00013 AC006247 174368 AC006247 121474 AC007150 129429 129429 931 36224 35881 83657 2339 405 AC004951 AC004951 A8006546 Z99 125 U00013 AC007401 X93514 AQ148714 Table 4 (continulid) Mycobacterium leprae cosmld 1-536. Mycobacterium leprae 61.573 Mycobacterium Ieprae cosmid Bi496. Mycobacterium Ieprae 61,573 Drosophila melanogasler chromosome 2 clone BACR48110 (D505) RPCI-98 DrosophIla molanogaster 37.105 48.1.10 map 49E6-49F8 strain y; cn bw sp, SEQUENCING IN PROGRESS 17 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR48110 (0505) RPCI-98 Drosophila melanogasler 37.105 48.1,10 map 49E6-49F8 strain y; cn bw sp, -SEQUENCING IN PROGRESS -,17 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR161313 (0597) RPCI-98 Drosophila melanogasler 38,728 16.P.13 map 49E-49F strain y; cn bw sp. -SEQUENCING IN PROGRESS-, 87 unordered pieces.

Home sapiens clone D.11022114, -SEQUENCING IN PROGRESS ~,14 Home sapiens 33.116 unordered pieces.

Home sapiens clone D.11022114, -SEQUENCING IN PROGRESS *,14 Home sapiens 33,116 unordered places.

Ephydatla fluviatilis mRNA for G protein a subunit 4, partial cds. Ephydatia fluviatilis 36,379 Mycobacterium leprae cosmid L536. Mycobacterium leprae 48,401 Mycobacterlum leprae cosmd 61496. Mycobacterium leprac 48.401 Home sapiens clone NHO15OiOO7. SEQUENCING IN PROGRESS 3 Homo sapiens 37,128 unordered pleces.

C.glulamicum betP gene. Corynebacterium 38,889 glulamnicum HS_-3136_-Al_-A03_-MR CIT Approved Human Genomic Sperm Library 0 Home Homo sapiens 34.321 sapiens genomlic clone Plate=3i36 Col=5 Row=A, gonomic survey sequence.

Bacillus firmus dppABC operon. dipeptlde transporter protein dppA gene, Bacillus firmus 38,072 partial cds, and dipeplide transporter proteins dppB and dppC genes, complete 04-DEC-1998 01-MAR.1994 2-Aug-99 2-Aug-99 20-Sep-99 12-Jun -98 12-Jun-98 23-Jun-99 04-DEC-I1998 01-MAR-i1994 26-Jun-99 8-Sep-97 08-OCT-i1998 1-Feb-97 01-MAR-1994 9-Apr-97 20-Aug-98 22-Jul-99 22-Jul-99 30-Jun-99 3837 U64514 36947 4032 41055 91421 91421 U00020 U85643 AL031317 AC0081 05 AC0081 05 Mycobacterium leprae cosmid B229.

Mycobacterium leprae 34,462 Pseudomonas syringae pv. syringae putativ'e dihydropteroate synthase gene, Pseudomonas syringae pv. 50,445 partial cds, regulatory protein MrsA (mrsA), triose phosphate Isomerase (tIA), syringae transport protein SecG (secG), (RNA-Leu. IRNA-Met, and 15 kDa protein genes, complete cds.

Streptomyces coelicolor cosmld 6G4. Streptomyces coelicolor 59,3 14 Home sapiens chromosome 17 clone 2020_-K_-17 map 17, -SEQUENCING Home sapiens 37,607 IN PROGRESS 12 unordered pieces.

Home sapiens chromosome 17 clone 2020_K_17 map 17, -SEQUENCING Home sapiens 37,607 IN PROGRESS 12 unordered pieces.

AVI 17143 Mus musculus C57BIJ6J 10-day embryo Mus musculus cDNA clone Mus musculus 40,157 2610200J 17, mRNA sequence.

GB-EST33:AV117143 222 AV1 17143 2007203039 29 Jun 2007 rxa02829 373 GB-HTG1:HSU9G8 GB-HTG1 :HSU9G8 GB-PR3:HSU85B5 rxc03216 1141 GB-HTG3:AC008184 48735 AL008714 48735 AL008714 39550 Z69724 151720 AC008184 GB-EST15:AA477537 411 AA477537 Table 4 (continued) Homo sapiens chromosome X clone LLOXNCO1-9G8. SEQUENCING IN Homo sapiens PROGRESS in unordered pieces.

Homo sapiens chromosome X clone LL0XNCOI-9G8. SEQUENCING IN Homo sapiens PROGRESS in unordered pieces.

Human DNA sequence from cosmid U85135, between markers DXS366 and .Homo sapiens DXS87 on chromosome X.

Drosophila melanogaster chromosome 2 clone BACRO4005 (D540) RPCI-98 Drosophila melanogaster 04.D.5 map 36E5-36F2 strain y: cn bw sp, SEQUENCING IN PROGRESS 27 unordered pieces.

zu36g12.rl Soates ovary tumor NbHOT Homo sapiens cDNA clone Homo sapiens IMAGE:740134 5 similar t0 contains Alu repetitive elemenl;conlalns element HGR repetitive element mRNA sequence.

fa91dO8.yl zebrafish fin dayl regeneration Danio rerio cONA mRNA Danio rerlo sequence.

Streptomyces coelicolor cosmId 3F9. Streplomyces coellcolor A3(2) SlIincolnensls (78-11) Lincomycin production genes. Streplomyces tincolnensis Homo sapiens chromosome 15 clone RP1 1-424J10 map 15, -SEQUENCING Homo sapiens IN PROGRESS 41 unordered pieces.

Homo sapiens chromosome 19, cosmid R30217. complete sequence. Homo sapiens Spombe chromosome I cosmid c926. Schizosaccharomyces pombe Archaeoglobus fulgidus section 26 of 172 of the complete genorne. Archaeoglobus fulgldus 41. 595 4 1.595 41,595 39,600 37,260 9-Nov.97 23-Nov-99 23-Nov-99 23-Nov-99 2-Aug-99 rxs03215 1038 rxs03224 1288 GB-EST26:A1330662 GBBA1:5C3F9 GBBA1:SLLINC GBHTG5:AC009660 GB-PR3:AC004076 GB-PL2:SPAC926 GSBA2:AEOO1O81 412 A1330662 19830 AL023862 36270 X79146 204320 AC009660 41322 AC004076 23193 AL110469 11473 AE001081 37,805 48,657 39,430 35,151 37,788 38,474 35,87 1 28-DEC- 1998 10-Feb-99 15-MAY-1996 04-DEC-1999 29-Jan-98 2-Sep-99 1 5-DEC-1997 -118- Exemplification Example 1: Preparation of total genomic DNA of Corynebacterium glutamicum ATCC 13032 A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 0 C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I of the original volume of the culture all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/1 sucrose, 2.46 g/l MgSO, x 7H 2 0, 10 ml/l KHPO, solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/1 M12 concentrate (10 g/1 (NH-) 2 SO,, I g/1 NaCI, 2 g/l MgSO, x 7HO, 0.2 g/1 CaCI,, 0.5 g/l yeast extract (Difco), 10 ml/1 trace-elements-mix (200 mg/l FeSO.

x H 2 O, 10 mg/l ZnSO, x 7 H20, 3 mg/1 MnCI, x 4 H,O, 30 mg/l H,BO, 20 mg/l CoCI, x 6 HO 2 1 mg/1 NiCI, x 6 H 2 0, 3 mg/1 Na 2 MoO, x 2 H 2 0, 500 mg/1 complexing agent (EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/l biotin, 0.2 mg/1 folic acid, mg/l p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/l ca-panthothenate, 140 mg/1 nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 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-HCl, 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 ofproteinase K to a final concentration of 200 ig/ml, the suspension is incubated for ca.18 h at 37 0 C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroformisoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -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 4g/ml RNaseA and dialysed at 4 0 C against 1000 ml TE-buffer for at least 3 hours.

During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added. After a min incubation at -20°C, the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer. DNA prepared by this proced,ure could be used for all purposes, including southern blotting or construction of genomic libraries.

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

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

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

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

783) or 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO. 784).

Example 4: In vivo Mutagenesis In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain -120the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.D.

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

Example 5: DNA Transfer Between Escherichia coli and Corynebacterium glutamicum Several Corynebacterium and Brevibacterium species contain endogenous plasmids (as pHMI519 or pBLl) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley Sons) to which a origin or replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E.

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

Using standard methods, it is possible to. clone a gene of interest into one of the shutle 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 (Kasts.umata, 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.

121 (1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough Murray (1983) J. Mol. Biol. 166:1-19).

Genes may be overexpressed in C. glutamicum strains using plasmids which comprise pCG1 Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, and A. J.

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

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

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

122- C (1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive c or chemiluminescent), such that when the total RNA of a culture of the organism is C 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 0 mutant gene. Total cellular RNA can be prepared from Corynebacteriumglutamicum by 0several methods, all well-known in the art, such as that described in Bormann, E.R. et al.

l' (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 123 advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NtLCI or NHOH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.

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

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

Culture conditions are defined separately for each experiment. The temperature should be in a range between 15C 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 124is also possible to maintain a constant culture pH through the addition of NaOH or NHLOH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the microorganisms, the pH can also be controlled using gaseous ammonia.

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

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

If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested. The medium is inoculated to an OD 600 of 0.5 1.5 using cells grown on agar plates, such as CM plates (10 g/l glucose, 2,5 g/1 NaCI, 2 g/l urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/l NaCI, 2 g/1 urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/1 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 125found, for example, in the following references: Dixon, and Webb, (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.

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

Press: Oxford; Boyer, ed. (1983) The Enzymes, 3 rd ed. Academic Press: New York; Bisswanger, (1994) Enzymkinetik, 2 nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, Bergmeyer, Gral1, eds. (1983-1986) Methods of Enzymatic Analysis, 3 rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim, p.

352-363.

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

The effect of such proteins on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar, H. et al. (1995) 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. glulamicum 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, 126- Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al.

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

(1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol..B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications.) In addition to the measurement of the final product of fermentation, it is .also possible to analyze other components of the metabolic pathways utilized for the production of the desired compound, such as intermediates and side-products, to determine the overall 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 127cells, then the cells are removed from the culture by low-speed centrifugation, and the supemate fraction is retained for further purification.

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

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

The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27- 32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540- 547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al.

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

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

USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 128- 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score 100, wordlength 12 to obtain nucleotide sequences homologous to SMP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score 50, wordlength 3 to obtain amino acid sequences homologous to SMP protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program XBLAST and NBLAST) for the specific sequence being analyzed.

Another example of a mathematical algorithm utilized for the comparison.of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci. 4: 11- 17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap.

length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, and include ADVANCE and ADAM. described in Torelli and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.

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

A comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the invention 129were compared to genes present in Genbank in a three-step process. In a first step, a BLASTN analysis a local alignment analysis) was performed for each of the sequences of the invention against the nucleotide sequences present in Genbank, and the top 500 hits were retained for further analysis. A subsequent FASTA search a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits. Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP program in the GCG software package (using standard parameters). In order to obtain correct results, the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4. The resulting data is identical to that which would have been obtained had a GAP (global) analysis alone been performed on each of the genes of the invention in comparison with each of the references in Genbank, but required significantly reduced computational time as compared to such a database-wide GAP (global) analysis. Sequences of the invention for which no alignments above the cutoff values were obtained are indicated on Table 4 by the absence of alignment information.

It will further be understood by one of ordinary skill in the art that the GAP alignment.

homology percentages set forth in Table 4 under the heading homology (GAP)" are listed in the European numerical format, wherein a represents a decimal point. For example, a value of "40,345" in this column represents "40.345%".

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

(1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label -130- CN may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification c of the relative or absolute amount of all or selected nucleic acids in the applied nucleic CN 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).

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

Nucleic acid m.icroarrays 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 131 described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).

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

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

Example 13: Analysis of the Dynamics of Cellular Protein Populations (Proteomics) The genes, compositions, and methods of the invention may be applied to study the interactions and dynamics of populations of proteins, termed 'proteomics'. Protein populations of interest include, but are not limited to, the total protein population of C.

glutamicum in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.

Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety 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: 132- 1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art.

Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors 35 S-methionine, 35 S-cysteine, 1 4 C-labelled amino acids, 1 5 N-amino acids, "NO3 or 15 NH4 or 1 3 C-labelled amino acids) in the medium of C. glutamicum permits the labeling of proteins from these cells prior to their separation. Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.

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

To determine the identity of any given protein, direct sequencing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degradation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, Langen 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.

133

EQUIVALENTS

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

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

Claims (30)

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:53, or a complement thereof.

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:54, or a complement thereof.

3. An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:54, 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:53, or a complement thereof. An isolated nucleic acid molecule comprising a fragment of at least contiguous nucleotides of the nucleotide sequence of SEQ ID NO:53, or a complement thereof.

6. An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:54, or a complement thereof.

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

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

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

11. The host cell of claim 10, wherein said cell is a microorganism. 135

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

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

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

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

16. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:54.

17. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:54.

18. An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:53.

19. An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:54. An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:54, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:54. 136

21. An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:53.

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

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

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

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

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

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

28. The method of claim 23, wherein said fine chemical is selected from the group consisting of: organic acids, proteinogenic and nonproteinogenic amino acids, purine and pyrimidine bases, nucleosides, nucleotides, lipids, saturated and unsaturated fatty acids, diols, carbohydrates, aromatic compounds, vitamins, cofactors, polyketides and enzymes. 137

29. The method of claim 23, wherein said fine chemical is an amino acid. The method of claim 29, wherein said amino acid is selected from the group consisting of: lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine, and tryptophan.

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

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

33. A host cell comprising the nucleic acid molecule of SEQ ID NO:53, wherein the nucleic acid molecule is disrupted.

34. A host cell comprising the nucleic acid molecule of SEQ ID NO:53, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:53. A host cell comprising the nucleic acid molecule of SEQ ID NO:53, wherein the regulatory region of the nucleic acid molecule is modified relative to the wild- type regulatory region of the molecule. BASF AKTIENGESELLSCHAFT WATERMARK PATENT TRADE MARK ATTORNEYS P20679AU03 Associated Physical Media Submitted III~ Basic Document (ie Conventi LIII Verified Translation LIZ Description Claims III] Abstract [III]Drawings ~III~ Gene Sequence Listing eLDRoo Other LI (eg. Deeds, Assignn on/Priority Document) ients, etc.)