AU2007203041A1 - Corynebacterium glutamicum genese encoding proteins involved in carbon metabolism and energy production - Google Patents

Description

-w P001 Section 29 Regulation 3.2(2)

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. I1, 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 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, Gierman 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.

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. gluramicum 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. glulamicum 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 detectiori 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. gluramicum 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.

-4- 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 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 due to the presence of a greater number of viable cells, each producing the 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 proteins, referred to herein as SMP proteins, which are capable of, for example, 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. 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:85, or a complement thereof.

An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:86, 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:86, or a complement thereof.

SAn isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:85, or a complement thereof.

An isolated nucleic acid molecule comprising a fragment of at least contiguous nucleotides of the nucleotide sequence of SEQ ID 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:86, or a complement thereof.

An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:86.

An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:86.

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 An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:86.

An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:86, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:86.

An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID A host cell comprising the nucleic acid molecule of SEQ ID wherein the nucleic acid molecule is disrupted.

A host cell comprising the nucleic acid molecule of SEQ ID wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID A host cell comprising the nucleic acid molecule of SEQ ID wherein the regulatory region of the nucleic acid molecule is modified relative to the wild-type regulatory region of the molecule.

Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding 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 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention a sequence of an oddnumbered SEQ ID NO in the Sequence Listing) 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 biologically active portion thereof.

Another aspect of the invention pertains to vectors, recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce 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 I 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.

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

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, 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 et al. (1998) Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.

A. Amino Acid Metabolism and Uses Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms. The term "amino acid" is 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: Weinheim, 1985.

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

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

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

Tryptophan is also produced from these two initial molecules, but its synthesis is an 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 ofaspartate. Isoleucine is formed from threonine. A complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-1-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 r d 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 Bl) is produced by the chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin B 2 is synthesized from (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively termed 'vitamin B 6 pyridoxine, pyridoxamine, pyridoxaand the commercially used pyridoxin hydrochloride) are all derivatives of the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic 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 1 2 and porphyrines belong to a group of chemicals characterized by a tetrapyrole ring system.

-16- The biosynthesis of vitamin B 1 2 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also termed 'niacin'. Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by largescale culture of microorganisms, such as riboflavin, Vitamin B 6 pantothenate, and biotin. Only Vitamin B12 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 r1 development of new drugs which can be used, for example, as immunosuppressants or Santi-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 n 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- S612). 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 turn, is converted to cytidine-5'-triphosphate (CTP).

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

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

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

II. 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: Peppler, H.J. and Perlman, D., eds. Microbial Technology 2 nd ed., vol. 2, chapter 18, Academic Press: New York; and 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 most important pathways in bacteria for sugar metabolism include the Embden- Meyerhoff-Pamas (EMP) pathway (also known as the glycolytic or fructose bisphosphate pathway), the hexosemonophosphate (HMP) pathway (also known as the 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 ofNADH, 1 molecule of acetyl-CoA, and 1 molecule of CO2. The acetyl group ofacetyl 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, oxaloacetate 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 oxygen or other terminal electron acceptors. This process is catalyzed by the respiratory chain, an electron transport system containing both integral membrane proteins and 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 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.

O 22- The action of the respiratory chain generates a proton gradient across the cell Smembrane, 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.

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

C Non-hexose carbon substrates may also serve as carbon and energy sources for Scells. Such substrates may first be converted to hexose sugars in the gluconeogenesis C 10 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 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 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 glulamicum 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 r various embodiments, the isolated SMP nucleic acid molecule can contain less than Sabout 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived a C. glutamicum cell). Moreover, an "isolated" nucleic acid molecule, such as a DNA molecule, can be substantially free of other cellular material, z or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

"1 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, N 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 all 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 1 -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 1 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.

eC¢ Additional nucleic acid fragments encoding biologically active portions of an C1 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 C 10 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 RXA00196 (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. gluramicum 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. glutamicum 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 -34another 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 O semi-conserved in the domain having SMP activity) may not be essential for activity and Sthus are likely to be amenable to alteration without altering SMP activity.

;Z 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 San 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 ri 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-2thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-

I

-38galactosylqueosine, inosine, N 6 -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 c-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual p-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids.

Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- -39methylribonucleotide (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-, lpp-, lac-, Ipp-lac-, lacIl-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SP02, X-PRor X PL, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, -41 usp, STLS1, 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. et al. (1991) "Heterologous gene expression in filamentous fungi" in: More Gene Manipulations in Fungi, J.W. Bennet L.L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C.A.M.J.J. Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium rumefaciens -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 pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the 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, pUC19, pKC30, pRep4, pHS pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- III 113-B1, Xgtl 1, pBdC1, 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 ld vector relies on transcription from a T7 gnl 0-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(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 pIJl01, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM 1519, pBL1, 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 pYepSec (Baldari, et al., (1987) Embo J. 6:229-234), 2 a, pAG-1, Yep6, Yepl 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 Sf 9 cells) include the pAc series (Smith et al.

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

In another embodiment, the 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+, O -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 O expression vector's control functions are often provided by viral regulatory elements.

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

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

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

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in O a manner which allows for expression (by transcription of the DNA molecule) of an

C

RNA molecule which is antisense to SMP mRNA. Regulatory sequences operatively Slinked 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 rC or attenuated virus in which antisense nucleic acids are produced under the control of a C high efficiency regulatory region, the activity of which can be determined by the cell 0 0 type into which the vector is introduced. For a discussion of the regulation of gene N 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 S(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 C, 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 C1 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 Sthe 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 CK1 recombinant DNA techniques, or chemical precursors or other chemicals when Schemically synthesized. The language "substantially free of cellular material" includes C 10 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 Sthe 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 T€ 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 Sis 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

C

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 Sthe 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 Cr 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 Sbiologically 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 m 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, C, 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-frame 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 et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the 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 S 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 O -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.

n D. Uses and Methods of the Invention (N 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 C 10 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; 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 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 8 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 Sthe 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 CO sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in C the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject. C. glutamicum and C. diphtheriae are C, related bacteria, and many of the nucleic acid and protein molecules in C. glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject.

The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies ofC. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutamicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the 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 Spermits 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 Cthe 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 C 10 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 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 -58- Sdue to the presence of a greater number of viable cells, each producing the desired fine Schemical.

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 Cc part of, for example, nucleotide molecules. The amount and efficiency of sugar Cmetabolism, 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. glulamicum 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.

2007203041 29 Jun 2007 TABLE 1: GENES IN THE APPLICATION H MP: Nucleic Acid SEQ ID NO 3 7

TCA:

Nucleic Acid SEQ ID NO 9 11 13 17 19 21 Amino Acid SEQ I0 NO 2 4 6 8 Amino Acid SEQ 10 NO 10 12 14 16 18 20 22 Identification Code RXS02735 RXA01626 RXA02245 RXA01 015 Identification Code R.XN01312 F RXAO 1312 R.XN0023 1 RXA0131 1 RXA01 535 RXA00517 RXA0 1350 Cip.

WV0074 GR00452 GROD654 GR00290 Conti!.

W0082 GR00380 WV0083 GR00380 GR00427 GROO131 GR00392 NT Start NT Stop Function 14576 4270 13639 346 15280 3926 14295 5 6-Phosphogiucotactonase L-ributose-phosphate 4-epimerase RIBULOSE-PHQSPHATE 3-EPIMERASE (EC 6.1.3.1) 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 SUB3UNIT (EC 1.3.99.1) SUCCINATE DEHYDROGENASE FLAVQPROTEIN SUBUNIT (EC 1.3:9.1) SUCCINATE-SEMIALODEHYDE DEHYOROGENASE (NADP+) (EC 1.2.1.16) SUCCINATE DEFIYDROGENASE IRON'-SULFUR PROTEIN (EC 1.3.99.11) FUMARATE HYDRATASE PRECURSOR (EC 4.2.1.2) MALATE DEHYDROGENASE (EC 1.1.1.37) (EC 1.1.1.82) MALATE DEHIYDROGENASE (EC 1. 1. 1.37) EMB-Pathway Nucleic Amino Acid Acid SEQ SEQ ID NO ID ND 23 24 26 27 28 29 30 31 32 33 3.4 36 tdentification Code RXA02 149 RXA01 814 RXN02803 F RXA02803 RXN03076 F RXA02854 RX.A0O11 Connig GR00639 GROO515 W0086 GR004 VV0043 GRIO002 GROG 129 NT Start NT Stop Function 17786 2571 2 1624 1588 18754 910 657 400 35 5 513 GLUCOKINASE (EC 2.7.1.2) PHOSPHOGLUCOMUITASE (EC 5.4.2.2)1 PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2) 1 PH-OSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (eC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUITASE (EC 5.4.2.2)1 PHOSPH-OMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2)1 PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUITASE (EC 5.4.2.2)1 PHOSPHOMANNOMUTASE (EC 5.4.2.8) 2007203041 29 Jun 2007 Table 1 (continued) Nucleic Acid Amino Acid Identific-ation Code Cui. NT Star NT StoF Function SEQ ID NO SEQ ID NO 37 38 RXN01365 WV0091 1476 103 PH-OSPHOGLUCOMUTASE (C 5.4.2.2) PHOSPHOMANNOMuTASE (EC 5.4.2.8) 39 40 F RXA01365 CR00397 897 4 PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) 41 42 RXA00098 CR00014 6525 8144 GLUOOSE-6-PHOSPHATE ISOMERASE (GPI) (EC 5.3.1.9) 43 44 RXA01989 CR00578 1 630 GLUCOSE-6-PHOSPHATE ISOMERASEA (GPI A)(EC 5.3.1.9) 46 RXAOO340 CR00059 1549 2694 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 47 48 RXA02492 GR00720 2201 2917 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 49 50 RXA00381 CR00082 1451 846 PHOSPHOCLYCERATE MUTASE (EC 5.4.2.1) 51 52 RXA02122 CR00636 6511 5813 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 53 54 RXA00206 CR00032 6171 5134 6-PHOS PHOFRUCTOKINASE (EC 2.7. 1.11) 56 R)(AO1243 CR00359 2302 3261 1-PHOSPHOFRUCTOKINASE (EC 2.7.1.56) 57 58 RXA01882 GR00538 1165 2154 1-PHOSPHOFRUCTOKINASE (EC 2.7.1.56) 59 60 RXA01702 CR00479 1397 366 FRUOTOSE-BISPHOSPHATE ALDOLASE (EC 4.1 .2.13) 61 62 RXA02258 CR00654 26451 27227 TRIOSEPHOSPHATE ISOMERASE (EC 5.3.1.1) 63 64 RXN01225 WV0064 6382 4943 GLYCERALOEHYDE 3-PHOSPHATE DEHYOROGENASE (EC 1.2.1.12) 66 F RXA01225 GR00354 5302 6741 GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE HOMOLOG 67 68 RXA02256 GR00654 23934 24935 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (EC 1.2.1.12) 69 70 RXA02257 CR00654 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 PYRU.VATE KINASE (EC 2.7.1.40) 77 78 F RXA02675 CR00754 2 364 PY RUVATE KINASE (EC 2.7.1.40) 79 80 F RXA02695 CR00755 2949 4370 PYRUVATE KINASE (C 2.7.1.40) 81 82 RXA00682 CR00179 5299 3401 PHOSPHOENOLPYRUVATE SYNTHASE (EC 2.7.9.2) 83 84 RXA00683 CR00179 6440 5349 PHOSPHOENOLPYRUVATE SYNTHASE (C 2.7.9.2) 86 RXN00635 WV0135 22708 20972 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC 1.2.2.2) 87 88 F RXA02807 CR00788 88 552 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC 1.2.2.2) 89 90 F RXA00635 CR00167 3 923 PYRUVATE DEHYDROGENASE (CYTOCHROME) (C 1.2.2.2) 91 92 RXN03044 WV0019 1391 2221 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 93 94 F RXA02852 CR00852 3 281 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 96 F RXA00268 CR00041 125 955 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 97 98 RXN03086 WV0049 2243 2650 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 99 100 F RXA02887 GR1 0022 411 4 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 101 102 RXN03043 W00O19 1 1362 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4.1) 103 104 F RXA02897 CR10039 1291 5 PYRUVATE DEHYDROGENASE El COMPONENT (C 1.2.4. 1) 105 106 RXN03083 WV0047 88 1110 DIHYDROLIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 107 108 F RXA02853 CR10001 69 1495 DIHYDROLIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 109 110 RXA02259 CR00654 27401 30172 PHOSPHOENOLPYRUVATE CARBOXYLASE (C 4.1.1.31) ill 112 RXN02326 WV0047 4500 5315 PYRUVATE CARBOXYLASE (EC 6.4. 1.1) 113 114 F RXA02326 CR00668 5338 4523 PYRUVATE CARBOXYLASE 115 116 RXN02327 WV0047 3533 4492 PYRUVATE CARBOXYLASE (ECO6.4.1.1) 117 118 F RXA02327 CR00668 6305 53.46 PYRUVATE CARBOXYLASE 119 120 RXN02328 WV0047 1842 3437 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 121 122 F RXA02328 CR00668 7783 6401 PYRUVATE CARBOXYLASE (EO 6.4.1.1) 123 124 RXN01048 WV0079 12539 11316 MALIC ENZYME (C 1.1.1.39) 2007203041 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ 10 NO 1 25 127 1 29 131 133 135 137 139 141 143 145 14 149 151 153 155 157 159 161 163 165 167 169 171 173 175 177 19 181 Amino Acid SEQ ID NO 126 128 130 132 134 136 138 140 142 144 146 148 150 152 154 156 158 160 162 164 166 168 170 172 174 176 178 180 Identification Code Cnr.

F RXA01048 F RXA00290 RXA02694 RXN00296 F RXA00296 RXA0 1901 RXN01 952 F RXA0 1952 F RXA01955 RXA00293 RXN0 1130 F RXA0130 RXNO 3112 F RXAOI1133 RXN00871 F RXA00871 RXN02829 F RXA02829 RXN0 1468 F RXA0 1468.

RXA00794 RXN02920 F RXA02379 RXN02688 RXN03087 RXN03186 RXN03187 RXN02591 RXS0 1260 RXS01 261 GR00296 GR00046 GR00755 W0176 GR00048 GR00544 WV0105 GR00562 GRO0562 GR00047 WO0157 GR00315 VV0085 GR00316 WO0127 GR00239 W0354 GR00816 V00 19 GR00422 GROO211 W02 13 GROOS90 W0098 W0052 W0377 WV0382 W0098 VVO0O9 W0009 3.

4693 1879 35763 3 4158 9954 4611 2645 6138 2 509 568 3127 2344 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 DEHYOROGENASE (CYTOCHROME) (EC 1.1.2.4) 0-LACTATE DEHYDROGENASE (CYTOCHROME) (C 1.1.2.4) L-LACTATE 0EHYDROGENASE (CYTOCHROME) (EC 1.1.2.3) 0-LACTATE DEHYOROGENASE (EC 1.1.1.28) 0-LACTATE DEHYDROGENASE (EC 1. 1. 1.25) 0-LACTATE DEHYDROGENASE (EC 1.1.1.28) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) D-3-PHOSPHOGLYCERATE DEHYDROGENAS E (EC 1. 1. 1.95) D-3-PHOSPHOGLYCERAtE DEHYDROGENAS$E (EC 1.1.1.95) D-3-PHOSPROGLYCERATE DEHYDIROGENASE (EC 1.1.1.95) IOLB. PROTEIN IOLB PROTEIN: 0-FRUCTOSE 1,6-BISPHOSPHATE GLYCERONE-CC PHOSPHATE +0D- GLYCERAILDEHYDE 3-PHOSPHATE.

IOLS PROTEIN IOLS PROTEIN NAGO PROTEIN PUTATIVE N-GLYCERALDEHYOE-2-PHOSPHOTRANSFERASE GLPX PROTEIN D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) 0-3-PHOSPHOGLYCERATE DEHYDROGENASE (E0 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 DEHYOROGENASE El COMPONENT (EC 1.2.4.1) PHOSPH-OENOLPYRUVATE CARBOXYKINASE (GTPj (EC 4.1.1.32) LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED- CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX (EC 1.8.1.4) LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED- CHAIN ALPHA-KETO ACID DEHYOROGENASE COMPLEX (EC 1.8.1.4) 287 287 7474 1250 3993 6135 1390 59053 3216 310 3 14370 3477 3703 183 184 Glycerol metabolism Nucleic Acid SEQ ID NO 185 187 189 191 193 195 Amino Acid SEQ ID NO 186 188 190 192 194 196 Identification Code Contig. NT Start NT Stop Function RXA02640 RXN0 1025 F RXAOI1025 RXA01 851 RXAO 1242 RXA02288 GR00749 WO0143 GR00293 GR00525 GR00359 GR00661 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 GLYCEIROL-3-PHOSPHATE DEHYDROGENASE (EC 1. 1.99.5) GLYCEROL-3-PHOSPHATE REGULON REPRESSOR GLYCEROL-3-PHOSPH-ATE REGULON REPRESSOR 2007203041 29 Jun 2007 Table I (continued) NT Start NT Stop Function Nucleic Acid Amino Acid Identification Code SEQ I0 NO SEQ ID NO 197 198 RXN01891 199 200 F RXA01891 Co9R.k W0122 24949 24086 GLYCEROL-3-PHOSPHATE-BINDING PERIPLASMIC PROTEIN

PRECURSOR

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

PRECURSOR

GR00703 3806 3062 Uncharacterized protein involved in glycerol metabolism (homolog of Drosophila rhomboid) W01 22 22091 22807 Clycerophosphoryl diester phosphodiesterase 201 202 203 204 RXA024 14 RXN01 580 Acetate metabolism Nucleic Acid Amino Acid Identification Code SEQ ID NO SEQ ID NO 205 206 RXA01436 207 208 RXA00686 209 210 RXA00246 211 212 R.XAO 1571 213 214 RXA01572 215 216 RXA01758 217 218 RXA02539 219 220 RXN03061 221 222 RXN03150 223 224 RXN01340 225 226 RXN01498 227 228 RXN02674 229 230 RXN00868 231 232 RXN01 143 233 234 RXN01 146 235 236 RXNO1 1144 ConrtR NT Start NT Stop Function CR00,418 2547 1357 ACETATE KINASE (EC 2.7.2.1) CR00179 8744 7941 ACETATE OPERON REPRESSOR CR00037 4425 3391 ALCOHOL DEHYDROGIENASE (EC 1.1.1.1) GR00438 1360 1959 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) CR00438 1928 2419 ALCOHOL DEHYDROGENASE (C 1.1.1.1) CR00498 3961 2945 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) GR00726 11676 10159 ALDEHYDE DEHYOROGENASE (EC WV0034 108 437 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) W0155 10678 10055 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) W0033 3 860 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) WV0008 1598 3160 ALDEHYDEODEHYDROGENASE (EC 1.2.1.3) WV031 5 15614 14163 ALDEHYDE DEHYOROGENASE. (EC 1.2.1.3) W0127 2230 320 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) WV0077 9372 8254 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) WV0264 243 935 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) W0077 8237 7722 ACETOLACTATE SYNTHASE SMALL SUBUNIT (EC 4.1.3.18) Butanediol, diacetyl and acetoin formation Nucleic Acid Amino Acid Identification Code Conhig SEQ ID NO SEQ ID NO 237 238 RXA02474 GR007 239 240 RXA02453 GR007 241 242 RXS01758 VV0i12~ NT Start NT Stop Function 15 8082 7309 (S,S)-butane-2.3-diol dehydrogenase (EC 1. 1. 1.76) 10 6103 5351 ACETOIN(DIACETYL) REDUCTASE (EC 1.1.1.5) 27383 28399 ALCOHOL DEHYDROCENASE (EC 1.1.1.1) 2007203041 29 Jun 2007 Table 1 (continued) H MP -Cy cie Nucleic Acid SEQ ID NO 243 245 247 249 Amino Acid Identification Coda SEQ ID NO 244 RXA02737 246 RXA02738 248 RXA02739 250 RXA00965 Contig NT Start NT Stop Function GR00763 3312 1771 GLUCOSE-6-PHOSPHATE 1-DEHYDROGENASE (EC 1.1.1.49) GR00763 4499 3420 TRANSALOOLASE (EC 2.2.1.2) GR00763 6769 4670 TRANSKETOLASE (EC 2.2. 1. 1) GR00270 1232 510 6-PHOSPHOGLUCONATE DEH-YDROGENASE, DECARBOXYLATING (EC 1.1.1.44) W0106 2817 1366 6-PHOSPHOGLUCONATE DEHYOROGENASE, DECARBOXYLATING (EC 1.1.1.44) GR00283 3012 4448 6-PHOSPHOGLUCONATE DEHYOROGENASE, DECARBOXYLATING (EC 1.1.1.44) 251 252 RXN00999 253 254 F RXA00999 Nucleotide sugar conversion Nucleic Acid Amino Acid Identitication Code SEQ ID NO SEQ IDNO 255 256 RXN02596 257 258 F RXA02596 259 260 F RXA026342 261 262 R.XA02572 26.3 264 RKA02485 RXA0 1216 RXA0 1259 RXA02028 RXAOI 262 RXAO1 377 RXA02063 RXN0001 4 F RXAOOO 14 RXA0 1570 RXA02666 RXA00825 Contig VV0098 GR00742 GR00749 GR00737 GR00718 GR00352 GR00367 GR00616 GR00367 GROO400 GR00626 VV0048 GROO002 GR00438 GR00753 GR00222 48784 5383 2 2345 2302 987 573 8351 3935 3301 8848 4448 427 7260 222 47582 489 5880 646 3445 1202 '130 998 7191 5020 4527 9627 5227 1281 6493 1154 UOP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UDP-GALA;CTOPYRANOSE MUTASE (EC 5;4.99.9) UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UOP-GLUCOSE 6-DEHYDROGENASE (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-I1 -PHOSPHATE ADENYLYLTRANSFERASE (EC 2.7.7.27) GL UCOSE-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-RIB[TOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC 2.7.7.40) DTOP-'GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) NT Start NT Stop Function Inositol and ribitol metabolism Nucleic Acid SEQ ID NO 287 Amino Acid Identification Code SEQ I0 NO 288 RXA01887 Conti&. NT Start NT Stop Function GR00539 4219 3209 MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) 2007203041 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucteic 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 RXN00013 F RXAOOOI3 RXA0 1099 RXN01 332 F RX'A0 1332 RXA01632 RYAO 1633 RXN01 406 RXN01 630 RXN00528 RXN03057 F RXA02902 RXA00251 VV0048 GR00002 GR00306 VV0273 GR00388 GR00454 GROO.454 VV0278 VV0050 VV0079 V'/0028 GR1 0040 GR00038 7966 3566 6328 579 552 2338 3380 2999 48113 23406 7017 10277 931 8838 4438 5504 4 4 3342 4462 1977 47037 22318 7688 10948 224 MYO-INOSITOL-1(OR 4)-MONOPHOSPHATASE 1 (EC 3.1 .3.25) MYO-INOSITOL- 1(OR 4)-MONOPHOSPHATASE 1 (EC 3.1.3.25) INOSITOL MONOPHOSPHATE PHOSPHATASE MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) MYO-INOSITOL 2-DEH-YDROGENASE. (EC 1.1.1.1 8) MYO-INOSITOL 2-DEHYOROGENASE (EC 1.1.1.1 8) MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) MYO*INOSITOL 2-DEHYDROGENASE (EC .1.1.1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC1.118 MYO-tNOSITOL-1 -PHOSPHATE SYNTH-ASE (EC 5.5.1.4) MYO-INOSITOL 2-DEHYDRO)GENASE (EC 1118 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) RIBtTOL 2-DEHYDROGENASE (EC 1.1.1.56) 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 RXN02 654 F RX(A02654 RXNO1O49 F RXA01049 F RXA01050 RXA00202 RXN00872 F RXA00872 RXN00799 333 334 F RXA00799 RXA00032 RXA02528 RXN0031 6 F RxAoo3o9 GR00752 VV0079 GR00296 GR00296 GR00032 W01 27 GR00240 W0009 GR00214 GR00003 GR00725 WV0006 GR00053 WV0006 GR00053 GR00007 CR00615 GR00626 12206 7405 9633 1502 1972 1216 6557 565 511477 12028 6880 7035 316 6616 735 1246 725 1842 13090 8289 11114 492 1499 275 5604 1086 56834 1584 10520 7854 8180 5 7050 301 5 6 349 NT Start NT Stop Function GLUCOSE 1-DEHYDROGENASE (EC 1.1.1.47) GLUCOSE 1-DEHYDROGENASE 11 (EC 1.1.1.47) GLUCONOKINASE (EC 2.7.1.12) GLUCONOKINASE (EC 2.7.1.12) GLUCONOKtNASE (EC 2.7.1.12) D-RtBOSE-BINDING PERIPLASMIC PROTEIN PRECURSOR FRUCTOKINASE (EC 2.7.1.4) FRUCTOKINASE (EC 2.7.1.4) PERIPLASMIC BETA-CLUCOStDASEBETA-XYLOSIDASE PRECURSOR (EC 3.2.1.21) (EC 3.2.1.37) PERIPLASMIC BETA-GLUCOSIOASEIBETA-XYLOSIDASE PRECURSOR (EC 3.2. 1.2 1) (EC 3.2.1.37) MANNITOL 2-DEI-YDROGENASE (EC 1.1.1.67) FRUCTOSE REPRESSOR Hypothetical Oxidoreductase (EC GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) GLUCOSE-- FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.2 8) GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) SUCROSE-6-PHOSPHATE HYOROLASE (EC 3.2.1.26) SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) 343 344 RXNOO31O 345 346 F RXAOO310 347 348 RXA00041 349 350 RYA02026 351 352 RXA02061 2007203041 29 Jun 2007 Table I (continued) Nucleic Acid Amino Acid Identification Code Coni. NT Start NT Stop Function SEQ ID NO SEQ ID NO 353 354 RXN01 369 WV0124 595 1776 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8) 355 356 F RYA01369 GR00398 3 503 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8) 357 358 F RXA01373 GR00399 595 1302 MANNOSE-6-PHOSPHATE ISOMERASE (EC 5.3.1.8) 359 360 RXA0261I 1 0R00743 1 1752 1,4-ALPHA-GLUCAN BRANCHING ENZYME (EC 2.4.1.18) 361 362 RXA02612 GR00743 1793 3985 1,4-A LPHA-GLUCAN BRANCHING ENZYME (EC 2.4.1.18) 363 364 RXN01 884 VVO 184 1 1890 GLYCOGEN DEBRANCHING ENZYME (PC 2.4.1.25) (EC 3.2.1.33) 365 366 F RX-A01884 GR00539 3 1475 GLYCOGEN DEBRANCHING ENZYME (EC 2.4.1.25) (EC 3.2.1.33) 367 368 RXA01I I 1 GR00306 16981 17427 GLYCOGEN OPERON PROTEIN GLGX (EC 369 370 RXN01 .550 VV0143 14749 16260 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 371 372 F RXAO 1550 GR00431 3 13U6 GLYC6GEN PHOSPHORYLASE (EC 2.4. 1.1) 373 374 RXN02100 WV0318 2 2326 GLYCOG.EN PHOSPHORYLASE (EC 2.4.1.1) 375 376 F RXA02100 GR00631 3 920 GLYCOGEN PHOSPHORYLASE (EC 2.4,.1.1) 377 378 F RXA02113 GR00633 2 1207 GLYdCOGEN PHOSPHORYLASE (EC; 2.4.1.1) 379 380 RXA02147 CR00639 15516 16532 ALPHA-AMYLASE (EC 3.2.1.1) 381 382 RXAO1 478 GR00422 10517 12352 GLUCOAMYLASE Cl AND G2 PRECURSOR (EC; 3.2.1.3) 383 384 RXA01888 GR00539 4366 4923 GLUCOSE-RESISTANCE AMYLASE REGULATOR 385 386 RXN01927 VVN0127 50523 49244 XYLuLoSE KINASE (EC 2.7.1.17) 387 388 F RXA01927 GR00555 3 1118 XYLULOSE KINASE (EC 2.7.1.17) 389 390 RXA02729 CR00762 747 4 RIBOKINASE (EC 2.7.1.15) 391 392 RXA02797 GR00778 1739 2641 RIBOKINASE (EC 2.7.1. 393 394 RYA02730 GR00762 1768 731 RIBOSE COERON REPRESSOR 395 396 RXA02551 CR00729 2193 2552 6-PHOSPHO BETA-GLUCOWIASE (EC 3.2.1.86) 397 398 RXAOI-325 CR00385 5676 5005 DEOXYRIBOSE-PHOSPHATE AUJOLASE (EC 4.1.2.4) 399 400 RKA00195 CR00030 543 1103 1-deoxy-O-xylulose 5-phosphate reductolsomnerase (EC; 1. 1. 401 402 RYA00196 GROO030 1094 1708 1 -deoxy-D-xylulo~e 5-phosphate reductoisomerase (EC 1. 1.1l.-) 403 404 RXNOIS562 VVOI9I 1230 3137 1 .DEOXYXYLULOSE-5-PHOSPHATE

SYNTHASE

405 406 F RXA01562 CR00436 2 1039 1.-DEOX5YXYLULOSE-56PHOSPHATE

SYNTHASE

407 408 F RYA01705 CR00480 971 1573 1 .dEO(YXYLULbSE-5.-PHOSP1ATE

SYNTHASE

409 410 RXN00879 WV0099 8763 6646 4.ALPHA-GLUCANOTRANSFERASE (EC,24.1.25).

411 412 FRA07 GR04 597 3828 4-ALPHA-CLUCANOTRANSFtRASE (EC 2:4.1.25), amylomakase 413 414 RXA0087 19 3042 524 287 ~CTLLCSMN--HSHT ECTLS E 415 4164 RXOOO43 R00007 3244 2081 N-ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE (EC, 3.5.1.25) 417 418 RXN01752 WV0127 35265 33805 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 419 420 F RXA01 839 GR00520 1157 5 10 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 421 422 RXA0 1859 CR00529 1473 547 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 423 424 RKA00042 GR00007 2037 1279 GLUCOSAMINE-6-PHOSPHATE ISOMERASE (EC 5.3.1.10) 425 426 RXA01482 GR00422 17271 15397 GLUCOSAMINE--FRUCTOSE-6-PHOSPHATE AMvINOTRANSFERASE (ISOMERIZING) (EC 2.6.1.16) 427 428 RXN03179 WV0336 2 667 URONATE ISOMERASE (EC 5.3.1.12) 429 430 F RXA02872 GRIO013 675 4 URONATE ISOMERASE, Glucuronale Isomerase (EC 5.3.1.12) 431 432 RXNO3 18O WV0337 672 163 URONATE ISOMERASE (EC 5.3.1.12) 433 434 F RXA02573 CR10014 672 163 URONATE ISOMERASE, Clucufonate Isomerase (EC 5.3.1.12) 435 436 RXA02292 CR00662 1611 2285 GALACTOSIDE 0-ACIETYLTRANSFERASE (EC 2.3.1.18) 437 438 RXA02666 CR00753 7260 6493 D-RIBITOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC 2.7.7.40) 439 440 RXA00202 CR00032 1216 27*5 D.RIBOSE-BINDING PERIPLASMIC, PROTEIN PRECURSOR 441 442 RXA02440 CR00709 5097 4258 D.RIBOSE-BINDING PERIPLASMIC PROTEIN PRECURSOR 2007203041 29 Jun 2007 Table 1 lcontinued) Nucleic Acid Amino Acid Identification Code Cng. NT Start NT Stop Function SEQ ID NO SEQ tD NO 443 444 RXN01569 VV0009 41086 42444 dTDP-4.IJEHYORORHAMNOSE REDUCTASE (EC 1.1.1.133) 445 446 F RXA01 569 GR00438 2 427 DTDP-4-DEH-YORORHAMNOSE REDUCTASE (EC 1.1.1.133) 447 448 F RXA02055 GR00624 7122 8042 OTDP-4-DEHYDRORHAMNOSE REDUCTASE (EC 1.1.1.133) 449 450 RXA00825 GR00222 222 1154 DTOP-GLUCOSE 4,6-b.EHYDRATASE (EC 4.2.1.46) 451 452 RXA02054 GR00624 6103 7119 OTDP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) 453 454 RXN00427 WV01 12 7004 6219 dTDP-RHAMNOSYL TR.ANSFERASE RFBF (EC 455 456 F RXA00427 GROO098 1591 2022 DTOP-RHAMNOSYL TRANSFERASE RFBF (EC 457 458 RXA00321 GR00057 10263 9880 PROTEIN ARAJ 459 460 RXA00328 GRo067 11147 10656 PROTEIN ARAJ 461 462 RXA00329 GR00057 12390 11167 PROTEIN ARAJ 463 464 RXN01554 WV0135 28686 .26545 GLUCAN ENDO- 1,3-BETA-GLUCOSI DASE Al PRECURSOR (EC 3.2.1.39) 465 466 RXN03015 WV0063 289 8 UDP-GLU COSE 6-DEHY DROGENASE (EC 1. 1. 1.22) 467 468 RXN03056 w0028 6258 6935 PUTATIVEf H*EXULOSE-6-PHOSPHATE ISOMERASE (EC 469b 470 RXN03030 WV0009 57006 56443 PEMPLASMIC BETA-GLUCOSIDASEJBETA-XYLOSIDASE PRECURSOR (EC 3.2.1.21) (EC 3.2.1.37) 471 472 RXN00401 WV0025 12427 11489 5-DEHYDRO-4-DEOXYGLUCARATE DEHYDRATASE (EC 4.2.1.41) 473 474 RXN02125 W06102 23242 22442 ALD0SE REDUCTASE (EC 1. 1. 1.21) 475 476 RXN00200 VVO18I1 1679 5116 arabinosyl transferase subunit B (EC 477 478 RXN0111'75 W0017 3968B 38303 PHOSPHO-2-DEHYDRO-3-DEOXYHEPtONATE ALDOLASE (EC 4.1.2.15) 479 480 RXN01376 WOO9 1 5610 4750 PUTATIVE GLYCOSYL TRANSFERASE WBIF 481 482 RXN01631 WO0050 47021 46143 PUTATIVE HEXULOSE-6-PHOSPHATE ISOMERASE (EC 483 484 RXN01593 WV0229 132741 12408 NAGD PROTEIN 485 86 XN0037 019 2039 2418 GALATOKNAS (EC2.71.6 485 86 XN0037 VO 97 2369 2141 GAACTKINAE (C 2..1.)0C 487 488 RXS00584 WV0323 5516 6640 PHOSPHO-2-DEHYDRO-3-DEOXYHEPTONATE ALDOLASE (EC 4.1.2.15) 489 490 RXS02574 BETA-HEXOSAMINIDASE A PRECURSOR (EC 3.2.1.52) 491 492 RXS03215 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99 .28) 493 494 'F RXAO1915 GR00549 1 1008 GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) 495 496 RXS03224 CYCLOMALTODEXTRINASE (EC 3.2.1.54) 497 498 F RXA~oo38 GR00006 1417 260 CYCLOMALTOQEXTRINASE (EC 3.2.1.54) 409 500 RXCO6233 protein Involved in sugar metabolism 501 502 RXCOO2-36 Membrane Lipoproteln Involved in sugar metabolism 503 504 RXC00271 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 RXCO0412 Amino Acid ABC Transporter ATP-Blnding Protein involved in sugar metabolism 511 512 RXCO0526 ABC Transporter ATP-Bindling Protein Involved In sugar metabolism 513 514 RX661004 Membrane Spanning Protein involved in sugar metabolism 515 516 RXCO1017 Cytosolic Protein Involved In sugar metabolism 517 518 RXCO1021 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 RXC01366 Cytosolic Protein Involved in sugar metabolism 525 526 RXC01372 Cytosolic Protein involved In sugar metabolism 2007203041 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 53.4 536 538 540 542 Identification Code Coi. NT Start NT Stop Function RXCO1 659 RXCO1 663 RXCO1 693 RXCO1 703 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 GR00709 825 268 Uncharacterized protein Involved in glycerol metabolism (homolog of Drosophila rhomboid) protein involved in sugar metabolism 543 544 TICA-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 RX.AO2175 RXA02621 RXN00519 F RX00521 RXN02209 F RXA02209 RXN0213 F RXA02213 RXA02056 RXAOI 745 RXA00782 RXA00783 RXN01695 F RXAO 1615 F RXA01695 RX.AOO290 RXN0 1048 F RXAO1O48 F RXA00290 RXN03 101 Conlig.

GROO641 GR 00746 WV0144 GROW133 W06304 GRO0648 WV0305 GR00649 GR00625 GR00495 GR00206 GR00206 W0 139 GR00449 GR00474 GRO0045 W0079 GR00296 GR00046 WV0066 WV0025 WV0025 10710 2647 5585 2 3 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 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 DEHYDROGENASE [NADPI (EC 1. 1. 1.42) ACONITATE HYDRATASE (EC 4.2.1.3) ACONITATE HYDRATASE (EC 4.2.1.3) ACOOITATE HYDRATASE (EC 4.2.1.3) ACONITATE HYDRATASE (EC 4.2.1.3) 2-OXOGLUTARATE DEHYDROGENASE El COMPONENT (EC 1.2.4.2) 01l-YDROLIPOAMIDE SUCCINYLTRANSFERASE COMPONENT (E2) OF 2.OXOGLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.61) SUCCINYL-OA SYNTH-ETASE ALPHA CHAIN (EC 6.2.1.5) SUCCINYL-COA SYNTHETASE BETA CHAIN (EC 6.2.1.5) L-MALATE DEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) L-MALATE QEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) L-MALATE DEHYDROGENASE -(ACCEPTOR) (EC 1.1.99.16) MALIC ENZYME(E-C 1.1.1.39) MALIC ENZYM.E (EC 1.1.1.39) MALIC ENZYME (EC'1. 1.1.39) MALIC ENZYME (EC 1. 1. 1.39) QIHYDROLIPOAMIDE SUCCINYLTRANSFERASE COMPONENT (E2) OF 2.OXOGLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.61) DIHYDROLIPOAMIDE SUCCINYLTRANSFERASE COMPONENT OF 2- OXOGLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.61) oxoglutarate semialdehyde dehydrogenase (EC NT Star NT Stop Function 585 586 RXN02046 587 588 RXN00389 2007203041 29 Jun 2007 Glyoxylate bypass Table 1 (continued) NT Start NT Stop Function Nucfeic Acid SEQ ID NO 589 591 593 595 597 599 Amino Acid Identification Code SEQ ID NO 590 RXN02399 592 F RXA02399 594 RXN02404 596 F RXA02404 598 RXA01089 600 RXAO866 Conuig W0176 CIR00699 W0O176 GR0O700 GR00304 GR00539 19708 478 20259 3798 3209 3203 18365 1773 22475 1663 3958 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) CLYOXYLATE-INDUCED PROTEIN GLYOXYLATE-INDUCED PROTEIN Methy lcitrate-pathway Nucleic Aci SEQ ID NO 600 601 603 605 607 609 611 613 615 617 619 621 623 Amino Acid Identification Code SEQ ID NO 602 RXN03117 604 IF RXA00406 606 F RxA~o514 608 RXA00512 610 RXA00518 612 RXA01077 614 RXN03144 616 F RXA02322 618 RXA02329 620 RXA02332 622 RXN02333 624 F RXA02333 626 RXA00030 Conikg WV0092 CR00090 GR001130 GR00130 GR00131 GR00300 W61 41 CR60668 CR00669 CR00671 WV0141 GR00671 GR00003 NT Start NT Stop Function 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylcitrate synthase (EC 4.1.3.31) 2-methylcitrate synthase (EC 4.1.3.31) 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylcitrate synthase (EC 4.1.3.31) methytisocitrate lyase (EC 4.1.3.30) mnethylisocitrate lyase (EC 4.1.3.30) LACTOYLOLUTATHIONE LYASE (EC 4.4.1.5) Methyl-Malonyl-CoA-Mutases Nucleic Acid SEQ ID NO 625 627 629 Amino Acid Identification Code SEQ ID NO 628 RXN00148 630 F RXA00148 632 RXA00149 WO0167 CR00023 CR00023 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) 2007203041 29 Jun 2007 Table I (continued) Others Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function SEQ ID NO SEQ ID NO 631 634 RXN00317 W0197 26879 27532 PHOSPHOGLYCOLATE PHOSP-ATASE (EC 3.1.3.18) 635 636 F RXA00317 GR00055 344 6 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) 637 638 RXA021 96 GRCO6AS 3956 3264 PHOSPHOGLYCOLATE 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 Cog. NT Start NT Stop Function SEQ 103 NO SEQ ID NO 641 64 2 RXN01744 W0174 2350 812 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 643 644 F RXA00055 GRO0008 11753 11890 CYTOCHROMEO0UBIQUINOL OXIDASE SUBUNIT I(EC 1.10.3.-) 6-45 646 F RXAO1 744 GR00494 2113 812 CYTOCHROME D3 UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 647 648 RXA00379 GROO082 212 6 CYTOCHROME C-TYPE BIOGENESIS PROTEIN CCDA 649 650 RXA00385 GR00083 773 435 CYTOCHROME C-TYPE BIOGENESIS PROTEIN CCDA 651 652 RXA01743 GR00494 806 6 CYTOCHROME D3 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 RXA01 919 GR00550 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 RXA02140 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) 665 666 RXA02144 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--NAD(+) REDUCTASE (EC 1. 18.1.3) 679 680 RXA00224 GRCOO32 24965 24015 ELECTRON TRANSFER FLAVOPROTEIN ALPHA-SUBUNIT 681 682 RXA00225 GR00032 25783 24998 ELECTRON TRANSFER FLAVOPROTEIN BETA-SUBUNIT 683 684 RXN00606 VV0192 11299 9026 NADH- DEH-YDROGENASE 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 13EHYDROGENASE I CHAIN M (EC 1.6.5.3) 689 690 F RXA00608 GROD160 2253 3017 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 691 692 RXA00913 GR00249 3 2120 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 693 694 RXAOO9O9 GR00247 2552 3406 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 695 696 RXA00700 GRO0182 846 43 NADH-UBIOUINONE OXIDOREDUCTASE CHAIN 2 697 698 RXN00483 W0086 44824 46287 NADH-UBIOUINONE OXIDOREDUCTASE 39 KD SUBUNIT PRECURSOR 2007203041 29 Jun 2007 Nucleic Acid SEQ ID NO 699 Amino Acid SEQ ID NO 700 Identification Code F RXA00483 RXA01 534 RXA00288 RXA02741 RXN02560 F RXA02560 RXA01 311 RXNO3O 14 F RXA0091I0 RXN01 895 F RXA01895 RXAOO7O3 RXN00705 F RXAOO705 RXN00388 F RXA00388 F RXA00386 Tab Conrig NT Sla GRO01 19 19106 GR00427 1035 GR00046 2646 GR00763 9585 W0l0l 9922 GR00731 6339 GR00380 1611 W0058 1273 GR00248 3 W0O117 955 GR00543 2 GR00183 2556 VV0005 611.1 GR00184 1291 W0025 2081 GR00085 969 GR00084 514 GR00259 1876 W01101 5602 20569 NADH-UBIOUINONE OXIDOREDUCTASE 39 KO SUBUNIT PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) 547 NAOH-DEPENDENT FMVN OXYDOREDUCTASE 1636 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 8620 QUINONE OXIDOREDUCTASE (EC 1.6.5.5) 10788 NADPH-FLAVIN OXIDOREDUCTASE (EC 1.6.99.-) 7160 NADPH-FLAVIN OXIDOREDUCTASE (EC 1.6.99..) 865 SUCCINATE DEHYDROGENASE IRON-SULFUR PROTEIN (EC 1.3.99.1) 368 NADH- DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 1259 Hydrogenase subunits 5 NAOH DEHYDROGENASE (EC 1.6.99.3) 817 DEHYDROGENASE 271 FORMATE DEHYDROGENASE ALPHA CHAIN (EC 1.2.1.2) 5197 FDHD PROTEIN 407 FDHD PROTEIN 3091 CYTOCHROME C BIOGENESIS PROTEIN CCSA 667 essential protein similar to cytoclirome c 5 RESC PROTEIN, essential protein similar to cytochrome c biogenesis protein 2847 putative cytochrome oxldase 6759 FLAVOHEMOPROTEIN DIHYDROPTERIDINE REDUCTASE (EC 1 .6.997) 3176 FLAVOHEMOPROTEIN 3373 GLUTATHIONE s-TRANSFERASE (EC 2.5.1.18) 3134 GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE (EC 11.2.1.1) 11025 QCRC PROTEIN, men aquinol:cytochrome c oxidoreductase 4 NADI- DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 33063 NADH-UBIQUINONE OXIDOREDUCTASE CHAIN 4 (EC 1.6.5.3) 2794 Hypothetical Oxidorductase 849 Hypothetical Oxidoreduclase 4010 Hypothetical Oxidoreduclase (EC 1. 'le I (continued) rt NT Stop Function 733 734 RXA00945 735 736 RXN02556 F RXA02556 RXA01 392 RXAOO800 RXA021 43 RXN03096 RXN02036 RXN02765 RXN02206 RXN02554 GR00731 GR00408 GR00214 GR00639 VV0058 W0O176 WV0317 W0302 Wolol1 2019 2297 2031 10138 405 32683 3552 1784 4633 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 RX)A0 1204 RXAO 1201 RXN01 193 F RXAOI 193 F RXA01203 Coig VVO 121 GR00345 GR00344 WV0175 GR00343 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) 2007203041 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 767 769 771 773 775 777 Amino Acid Identific-ation Code Contig SEQ ID NO 768 RXN0282i WV01 2 770 F RXA02821 GRO08 772 RXA01200 GROOa 774 RXA01194 GRO03 776 RXA01 202 GRO03 778 RXN02434 W009( 1 324 02 139 44 2 43 770 44 2375 0 4923 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-BINDING PROTEIN Cytochrome metabolism Nucleic Acid Amino Acid Identification Code Conuig SEQ ID NO SEQ ID NO 779 780 RXN00684 WV0005 781 782 RXN00387 WV0025 NT Start NT Stop Function 29864 28581 1150 2004 CYTOCHROME P450 116 (EC 1. Hypothetical Cytochrome c Biogenesis Protein 2007203041 29 Jun 2007 2 Excluded Genes GcnBankIu 1 Gene Name Gene Function Reference Accession No. 1 A09073 ppg Phosphoenol pyruvate carboxylase Bachnmann, B. et al. "DNA fragment coding for phosphoenolpyruvat corboxylase, recombinant DNA carrying said fragment, strains carrying the recombinant DNA and method for producing L-aminino acids using said Patent: EP 0358940-A 3 03/21/90 A45579, Threonine dehydratase Moeckel, B. et "Production of L-isoleucine by means of recombinant A4558 1, micro-organisms with deregulated threonine dehydratase," Patent: WO A45583, 95 19442-A 5 07/20/95 A45585 ABOO3 132 murC; ftsQ; ftsZ Kobayashi, M. et "Cloning, sequencing, and characterization of the ftsZ gene from coryneform bacteria," Biochem. Biophzys. Re. Comm un., 236(2):383-388 (1997) ABO 15023 murC; flsQ Wachi, M. et al. "A murC gene from Coryneform bacteria," Appi. MicrobioL.

Bioi'echnol., 51(2):223-228 (1999) ABO 18530 dtsR Kimura, E. et "Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium Biosci. Biof'echnoI. Biochem., 60(10):1 565- 1570 (1996) AB018531 dtsRlI; 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 AB'027715 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 2007203041 29 Jun 2007 Table_2 cont AF0366O)I fdciAE; apt; rei AF04 1436 7F04 5998 AF047-64 AF049897 AF050 109 AF050 166 AFP051846 argR impA argH argC; argi; argB; argD); argF; argR; argG; argH inhA h isG hisA Dipeptide-bndigpotein; denin-e phosphoribosyltransferase;

GTP

pyrophosphokinase Arginine repressor Inositol monophosphate phosphatase Argininosuccinate lyase N-acetylglutamylphosphate reductase; ornithine acetyltransferase; Nacetylgiutamnate kinase; acetylomithine transminase; ornithine carbamoyltransferase; arginine repressor; argininosuccinate synthase; argininosuccinate lyase Enoyl-acyl carrier protein reductase ATP phosphoribosyltransferase Phosphoriljosylformimino5.-aminoo-

I-

phosphoribosyl-4-im idazolecarboxam ide isomerase Homoserine 0-acetyltransferase iud) I Wehmeier. L. et al. The role of the Corynebacterin~ glufarnicum. re! gcnc ;11n Park, S. et al. "Isolation and analysis of metA, a methionine biosynthetic gene encoding homoserine acetyltransferase in Corynebacterium glutamicum," Mo!.

Cells., 8(3):286-294 (1998) AF052652 1 metA AF053071i aroB ::IDehydroquinate synthetase

I

AF060558 I hisH Glutamine amidotransferase ni-n I I Au-vooliuq Phospfloribosyl-ATPpyrophosphohvdrolase AF 1 14111 i -A j 5-enouipyuvyM1Jkimate .J-pflospfate synthase Afl1~ZbOA 1 t. I Ar I 16 184 pan u L-aspartate-alptla-decarboxy lase precursor Dusch, N. et al. "Expression o he Corynebacterium glutamicum pa'nD geneencoding L-aspartate-alpha-decarboxylase leads to pantothenate overproduction in Escherichia coli," App!. Environ. Microbiol., 65(4)1530- 1539 (1999) AF12ASi12 1 -r)f -r 1 1 I i dehydrogenase t I

I

tfV IZ4UVV pepQ ro;aro Chorismate synthase; sh ikimate kinase; 3dehydroquiniate synthase; putative AF145897 inhAI_ AF145898 2007203041 29 Jun 2007 2 (continued) A.1001436 ectP ITransport of ectoine, glyci ne betaine, Peter, H. et "Corynebacterium glutamicumn is equipped with four secondary Iproline Icarriers for compatible solutes: Identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine carrier, EctP," J Bacterial., 180(22):6005-6012 (1998) AJO04934 dapD Tetrahydrod ipicol in ate succinylase Wehrmann, A. et "Different modes of diaminopimelate synthesis and their (incompletel) role in cellI wall integrity: A study with Corynebacterium glutamicum," J 180(1 2):3 159-3165 (1998) AJO07732 ppc; secG; am(; ocd; Phosphoeno lpyruvate-carboxy lase; high soxA affinity ammonium uptake protein; putative omnithine-cyclodecarboxylase; sarcosine oxidase AJO 103 19 fisY, gloB, gInD; smp; involved in cell division; PH1 protein; Jakoby, M. et "Nitrogen regulation in Corynebacterium glutamicum; amtP uridylyltransferase (uridylyl-removing Isolation of genes involved in biochemical characterization of corresponding enz-mye); signal recognition particle; low proteins," FEMS Microbial., 173(2):303-310 (1999) affinity ammonium uptake protein AJ132968 cat Chloramphenicol aceteyl transferase AJ224946 mqo L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Coryncbactcrium 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) D1 7429 Transposable element 1S3 1831 Vertes et al."Isolation and characterization of IS3 183 1, a transposable element from Corynebacterium glutamicum," Mal. Microbial., 1] (4):739-746 (1994) D84 102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al. "Molecular cloning of the Corynebacterium glutamicumn (Brevibacterium lactofermentum AJ 12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase," Microbialagy, 142:3347-3354 (1996) E01358 hdh; hk 1-omoserine dehydrogenase; homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: P_ kinase 1987232392-A 1 10/12187 E01359 upstream of the start codon of homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent: JP kinase gene 1987232392-A 2 10/12/87 E0 1375 Tryptophan operon E01376 trpL; trpE Leader peptide; anthranilate synthase Matsui, K. et al. "Tryptophan operon, peptide and protein coded thereby, utilization of tryptophan operon gene expression and production of Patent: JP 1987244382-A 1 10/24/87 2007203041 29 Jun 2007 2 (continued)I EQ01377 [IPromoter and operator regions of I Matsui, K. et al. "Trvntonhan operon, peptide and protein roded therehy, 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 biotin synthetase and its utilization," Patent: JP 1992278088-A 1 10/02/92 £0fO4040 Diamino pelargonic acid aminotransferase Kohama, K. et al. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A 1 E04041I Desth iobilot insynthctase Kohamna, K. et al. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthctase and its utilization," Patent: JP 1992330284-A I 111.18/02 E04307 Flavum aspartase Kurusu, Y. et al. "Gene DNA coding aspartase and utilization thereof," Patent: 1993030977-A I102/09/93 E04376 Isocitric acid lyase Katsumata, R. et al. 'Gerie manifestation controlling DNA," Patent: JP 3 03/09/93 E04377 Isocitric acid lyase N-terminal fragment Katsumata, R. et al. "Gene rnanifestdtion controlling DNA," Patent: JP 1993056782-A 3 03/09/93 E04484 Prephenate dehydratase Soiouchi, N. et al. "Production of L-phenylalanine by fermnentation," Patent: JP 1993076352-A 2.03/30/93* 108 Aspartokinase Fugono, N. et-al. "Gene DNA coding Aspartokinase and its use," Patent: JP 1993184366-A 1 07/27/93 EQS5112 Dihydro-dipichorinate synthetase Hatakeyamna, K. et al. "Gene DNA coding dihydrodipicolinic acid synthetase and its use," Patent: JIP 1993184371-A 1 07/27/93 E05776 Diaminopimelic acid dehydrogenase Kobayashi, M. et al. "Gdne DNA. coding Diaminopimelic acid dehydrogenase its use," Patent:. JP1 993294910-A '1 11/0/93 E05779 Threonine synthase Kohama, K. et al. "Gene-DNA coding threonine synthase and its use," Patent: 1993284972-A 1 1-1/02/93 E061 10 Prephenate dehydratase Kikuchi, T. et al. "Production of L-phenylalanine by fermentation method," Patent: JP 199334488 1-A 1 12/27/93 E061 I I Mutated Prephienate dehydratase Kikuchi, T. et al. "Production of L-phenylalanine by fermentation method," JP 1993344881awA 1 12/27/93 E06146 Acetohydroxy acid synthetase lnui, M. et al. "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: JIP 1994062866-A I 03/08/94 2007203041 29 Jun 2007 E06827 I 2 (continued) e l Mtn saikns aet E0682 Mutated aspartokinase alpha subunit Simoto, M. e L"uatsprois gene," paetIP 1994062866-A I E07701 secY Honno, N. et al. "Gene DNA participating in integration of membraneous to membrane," Patent: JP 1994169780-A 1 06/21/94 E08 177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from inhibition and its utilization," Patent: .IP 1994261766-A 1 09/20/94 E08 178, Feedback i nh ibiti on-re leased Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from E08179, feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08 180, E08 18 1, E08 182 £08232 Acetohydroxy-acid isomeroreductase lnui, M. et al. "Gene DNA coding acetohydroxy acid isomeroreductase," Patent:,J P.1 994277067-A-1 1 0/0494..- £08234 secE As'ai, Y. et al. "Gene DNA codinigT -or translocation machinery of protein," JP 1994277073-A 1 10/04/94 £08643 FT aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA fragment having promoter function in promoter region coryneform bacterium," Paten(: JP 199503 1476-A 1 02/03/95 E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having promoter function in bacterium," Patent: JP 199503 1476-A 1 02/03/95 E08649 Aspartase Kohamna, 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 1 03/20/95 £08901 Diaminopimelic acid decarboxylase Madori, M. et al. "DNA fragment containing gene codin g Diaminopimelic acid decarboxylase and utilization thereof," Patent: JP 1995075579-A 1 03/20/95 E12594 Serine hydroxymethyltransferase H-atakeyama, 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 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: JIP 1997070291-A 03/18/97 E12770 aspartokinase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 £12773 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 2007203041 29 Jun 2007 2 (continued) F 13655 1 T fliir.o,;e-A.nho-nhafp e.i-hvciror'ennp Hfntk(rv~mn W et qI "(iIhirn--nhncnh~tp- rPhv rrropnnp Pnri fNA rnnkl coding the same," Patent: JP 199722466 1 -A 1 09/02/97 L01508 iivA Threonine dehydratase Moeckel, B. et al. "Functional and structural analysis of the threonine dehydratase of Corynebacterium glutamicum," J. Bacterial, 174:8065-8072 L07603 EC 4.2.1.15 3-deoxy-D-arabinoheptulosonate-7- Chen, C. et al. "The cloning and nucleotide sequence of Corynebacterium phosphate synthase glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene," FEMS Microbial Left., 107:223-230 (1993) L09232 llvB; ilvN; ilvC Acetohydroxy acid synthase large subunit; Keilhauer, C. et al. "Isoleucine synthesis in Corynebacterium glutamicum: Acetohydroxy acid synthase small subunit; molecular analysis of the ilvB-ilvN-ilvC operon," J Bacterial, 175(!7):5595- Acetohydroxy acid isomeroreductase 5603 (1993) L18874 PtsM Phosphoenolpyruvate sugar Fouet, Acet al. "Bacillus subtilis sucrose-specific enzyme 1! of the phosphotransferase phosphotransferase system: expression in Escherichia coli and homology to enzymes 11 from enteric bacteria," PNAS USA, 84(24):8773-8777 (1987); Lee, J.K. et al. "Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme .11 and analyses of the deduced protein sequence," FEMS Microbial. Let., 1 19(1-2):137-145 (1994) L27 123 aceB Malate synthase Lee, H-S. et "Molecular characterization of aceB, a gene encoding malate synthase in Corynebacterium glutamicum," J. Microbial. Biotechnol., 4(4):256-263 (1994) L27 126 Pyruvate kinase Jetten, M. S. et "Structural and functional analysis of pyruvate kinase from Corynebacterium glutam icum," Appi. Environ. Microbial., 60(7):250 1-2507 L28760 aceA Isocitrate lyase IL35906 dtxr Diphtheria toxin repressor Oguiza, J.A. et "Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae.dtxR from Brevibacteriumn J Bacterial., 1 77(2):465-467 (1995) M 13774 Prephenate dehydratase Follettic, M.T. et "Molecular cloning and nucleotide sequence of thle ____________Corynebactenium glutamicum pheA gene," J. Bacterial., 167:695-702 (1986) M 16175 5S rRNA Park, Y-H. et al. "Phylogenetic analysis of the coryneform bacteria by 56 rRNA sequences," J Bacterial., 169:180-1-1806 (1987) M 16663 trp2 Antliranilate synthase, 5' end Sano, K. et "Structure and function of the trp operon control regions of Brevibacterium lactofermentum, a glutamic-acid-producing bacterium," Gene, 52:191-200 (1987) M 16664 trpA Tryptophan synthase, 3'end Sano, K. et al. "Structure and function of the trp operon control regions of Brevibacterium lactofermentum, a glutamnic-acid-producing bacterium," Gene, 52:191-200 (1987) 2007203041 29 Jun 2007 Table 2 (continued I M25819 1fPhosphoenolpyruvate carboxylase 1O'Rcgan, M. et al. "Cloning and nucleotide sequence of the I I IPhnosnhenolnvtn' rsrboxylaqe-rnding gene of Corynebacterium glutamicum ATCC 13032," Gene. 77(2):237-251I (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-I1175 (1992) 107, 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive bacteria with a high DNA G+C content are 108 characterized by a common insertion within their 23S rRNA genes," J Gen.

Microbial, 138:1167-1175 (1992) M89931 aecD; brnQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, 1. et al. "The Corynebacterium glutamicumn aecD gene encodes a C-S uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination activity that degrades aminoethylcysteine," J Bacleriol, 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the bmnQ gene Arch. Microbial, 169(4):303-312 (1998).

S59299 trp Leader gene (promoter) Herry, D.M. et al. "Cloning of the trp gene cluster from a tryptophanhyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence," Appi. Environ. Microbial, 59(3):791-799 (1993) UI 11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican, L.K. (1994) Complete nucleotide sequence of the Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology Department, University College Galway, Ireland.

U 13922 cg!IM, cgllR; c~glIR Putative type!!1 5-cytosoine Schafer, A. et al. "Cloning and characterization of a DNA region encoding a m ethyl transferase; putative type 11 stress-sensitive restriction system from Corynebacterium glutamicumn ATCC restriction endonuclease; putative type I or 13032 and analysis of its role in intergeneric conjugation with Eseherichia type IIl restriction endonuclease coli," J. Bacieriol., 176(23):7309-7319 (1994); Schafer, A. et "The Corynebacterium glutamicum eglIM gene encoding a 5-cytosine in an McrBCdeficient Eseherichia coli strain," Gene, 203(2):95-101 (1997) U 14965 recA U3 1224 px Ankri, S. et "Mutations in the Corynebacteriumn glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J Iacteriol., 178(15):44*12-4419 (1996) U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et "Mutations in the Corynebacteriumnglulamicumproline biosy nthetic pathway: A natural bypass of the proA step," J Baclerial, 178(15):4412-4419 (1996) U3 1230 obg; prol3; unkdh ?;gammna glutamyl kinase;similar to D- Ankri, S. et "Mutations in the Corynebacteriumn glutamnicumproline isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the proA step," J Bacterial, 178(15)44 12-4419 (1996) 2007203041 29 Jun 2007 Table 2 (continued) I IJ3281 IbioBI Rioin svthaI Serebriiskii, IC0., "Two new members of the bio B superf;.mily: Cloning sequencing and expression of bio B genes of Methylobacillus flagellatum and Corynebacterium glutam icum," Gene, 175: 15-22 (1996) U35023 thtR; accBC Thiosulfate sulfurtransferase; acyi CoA Jager, W. et al. "A Corynebacterium glutamnicum gcne encoding a two-domain carboxylase protein similar to biotin carboxylases and biot in -carboxyl -carrier proteins," Arch. Microbiol., 166(2);76-82 (1996)' U43535 cmr Multidrug resistance protein Jager, W. et at. "A Corynebacteriumn glutamicum gene conferring multidrug resistance in the heterologous host Escherichia coli," J Bacteriol., 179(7):2449-2451 (1997) U43536 clpB Heat shock ATP-binding protein U53587 aphA-3 3'S' -am inoglycoside phosphotransferase U89648 Coryncbacterium glutamicum unidentified sequence involved in histidine biosynthesis, partial sequence X04960 trpA; trpB; trpC; trpD; Tryptophan operon Matsui, K. et al. "Complete nucleotide and deduced amino acid sequences of trpE; trpG; trpL the Brevibacterium lactofermentum tryptophan operon," Nucleic Acids Res., 14,(24):101 13-10114 (1986) X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al. "Nucleic sequence of the lysA gene of Corynebacterium decarboxylase, EC 4.1.1.20) glutamicum and possible mechanisms for modulation of its expression," Mo.

Gen. Genet., 212(1):1 12-1 19 (1988) X14234 EC 4.1.1.31 Phosphoenolpyruvate carboxylase Eikmanns, B.J. et at. "The Phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and expression," Mo. Gen. Gene., 21 8(2):330-339 (1989); Lepiniec, L. et at.

"Sorghum Phosphoenolpyruvate carboxylase gene family: Structure, function and molecular evolution," Plant. Mo! Bio., 21 (3):487-502 (1993) X173 13 fda Fructose-bisphosphate aldolase Von der Osten, C.H. et at. "Molecular cloning, nucleotide sequence and finestructural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose-I1, 6-biphosphate aldolase to class I and class 11 aldolases," Mo!. Microbiol., X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al. "Nucleic sequence of the dapA gene from Corynebacterium glutamicum," Nucleic Acids Res., 18(21):6421 (1990) X54223 AttB-related site Cianciotto, N. et at. "DNA sequence homology between att B-related sites of Corynebacteriumn diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum and the attP site of lambdacorynephage," FEMS. Micro biol, Lett., 66:299-302 (1990) X54740 argS; lysA Arginyl-tRNA synthetase; Diaminopimelate Marcel, T. et al. "Nucleotide sequence and organization of the upstream region decarboxylase of the Corynebacterium glutamicum lysA gene," Mo!. Microbiol., 4(1 l):l8 19- 11830 (1990) 2007203041 29 Jun 2007 Table 2 (continued) X55994 trpL; irpE Putative leader peptide; anthranilate Heery, D.M. et al. "Nucleotide sequence of the Corynebacterium glutamicum Isynthase cornponerL I rpE gene," AlucleicAcid.- R. 181)7 3(8O11090 X56037 thrC Threonine synthase Han, K.S. et al. "The molecular structure of the Corynebacterium glutamicum threonine synthase gene," al. Microbial, 4(10):1693-1702 (1990) X56075 attB-related site Attachment site Cianciotto, N. et al. "DNA sequence homology between att B-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum and the attP site of lambdacorynephage," FEMS. Microbial, Left., 66:299-302 (1990) X57226 lysC-alpha; lysC-beta; Aspariokinase-aipha subunit; Kalinowski, J. et al. "Genetic and biochemical analysis of the Aspartokinase asd Aspartokinase-beta subunit; asparlate beta from Corynebacterium glutamicumn," Mo. Microbial, 5(5):1 197-1204 (1991); semnialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase genes lysO alpha and lysC beta overlap and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in glutamicum," Ma!. Gen. Genes., 224(3):317-324 (1999) X59403 gap;pgk; tpi Glyceraldehyde-3-phosphate; Eikmanns, "Identification, sequence analysis, and expression of a phosphoglycerate kinase; triosephosphate Corynebacterium glutamicum gene cluster encoding the three glycolytic isomerase enzymes .glyceraldehyde-3 -phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomeras," J. Bacterial, 174(19):6076-6086 X59404 gdh Glutamate dehydrogenase Bormann, E.R. et al. "Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase," Ma!. Microbial, 6(3):317-326 (1992) X60312 lysi L-lysine permease Seep-Feldhaus, A.H. et al. "Molecular analysis of the Corynebacteriumn glutamicum lys] gene involved in lysine uptake," AM. Microbial., 5(12):2995- 3005 (1991) X66078 cop I Psi protein Jouff, G. et al. "Cloning and nucleotide sequence of the csplI gene encoding PSI, one of the two major secreted proteins of Corynebacterium glutamicum: The deduced N-terminal region of PSI is similar to the Mycobacterium antigen complex," MoL Microbial, 6(1 6):2349-2362 (1992) X66 112 gut Citrate synthase Eikmanns, 8.1. et al. "Cloning sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gItA gene encoding citrate synthase," Microbial, 140:1817-1828 (1994) X67737 dapB Dihydrodipicolinate reductase X69 103 csp2 Surface layer protein PS2 Peyret, J.L. et al. "Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum," Mo. Microbial, 9(l):97-109 (1993) X69 104 IS3 related insertion element Bo'namy, C. et al. "Identification of IS 1206, a Corynebacterium glutamicum 1S3-related insertion sequence and phylogenetic analysis," Mol. Microbial, 14(3):571-581 (1994) 2007203041 29 Jun 2007 teuA Table 2 (continu 1-AIsopropyimalate synthase P-atek, M. et at. "Leucine synthesis in Corynebacterium glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis," App!. Environ. Microbiol, 60(t):133-140 (1994) X_ 71489 icd Isocitrate dehydrogenase (NADP+) Eikmanns, B.i. et Ml. "Cloning sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme,"JI Bacieriol, 177(3):774-782 (1995) X72855 GDHA Glutamate dehydrogenase (NA DP+) X 7 5 083, mtrA 5-methyltryptophan resistance Heery, D.M. et al. "A sequence from a tryptophan-hyperproducing strain of X70584 Corynebacterium glutamicumn encoding resistance to Biochem. Biophys. Res. Commun., 201(3):1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA mutant strains of Gorynebacterium glutamicum and Brevibacteriumn lactofermentum," App!.

Biolechnol. 42(4):575-580 (1994) X75504 aceA; thiX Partial Isocitrate lyase; Reinscheid, D.J. et al. "Characterization of the isocitrate lyase gene frm Corynebacterium glutam icum and biochemical analysis of the enzyme,"J Bacteriol., 176(12):3474-3483 (1994) X76875 ATPase beta-subunit Ludwig, W. et al. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes," Antonie Van Lceuivenhoek 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 genes," Anronie Van Leemvenhoek 64:285-305 (1993) X77384 recA Billman-Jacobe, H. "Nucleotide sequence of a recA gene from Corynebacterium glutamicum," DNVA 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," 140:3099-3 108 (1994) X80629 16S rDNA 16S ribosomnal 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," Microbiol., 14 1:523-528 X8 1191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, WV. et al. "Structure of the gluABCD cluster encoding the gln D glutamate uptake system of Corynebacterium glutam icum," J Bacteriol., 177(5):1 152-1158 (1995) X& 1379 dapE Succinyldiaminopimelate desuccinylase Wehrmnann, A. et al. "Analysis of different DNA fragments of Corynebacterium glutamicum complementing dapE of Escherichia coli," 40:3349-56 (1994) 2007203041 29 Jun 2007 X8206l l S rD N A 16S ribo omal R N AT ab le 2 (con tin u ed )a.o thde u d X8261 6SrDN 16 rbosmalR1 Ruimy, R. et al Phylogeny oftegenus Corynebacterium deucdfrom.

45(4):740-746 (1995)1 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.

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

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

glutamicum proline reveals the presence of aroP, which encodes the aromatic acid transporter," J. Bacteriol., 177(20):5991-5993 (1995) X86 157 argB; argC; argD; Acetylglutamnate kinase; N-acetyl-gamma- Sakanyan, V. et a1. "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 N- X89084 pta; ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D.J. et al. "Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutam icum pta-ack operon encoding ________________phosphotransacetylase and acetate kinase," Microbiology, 145:503-513 (1999) X89850 attB Attachment site Le Marrec, C. et al. "Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus C70," J. Bacteriol., 178(7):1996-2004 (1996) X90356 Promoter fragment Fl Patek, Mv. 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 FI10 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90359 Promoter fragment F13 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) 2007203041 29 Jun 2007 Table 2 (continued) A90360 Promoter fragment F22 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology.

142:1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et al. "Prrooters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90362 Promoter fragment F37 Patek, M. et al. "Promoters from C. glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90363 Promoter fragment 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) X90365 Promoter fragment F75 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90366 Promoter fragment PFI01 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90367 Promoter fragment PF 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 PF 109 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X93513 amt Ammonium transport system Siewe, R.M. et al. "Functional and genetic characterization of the (methyl) ammonium uptake carrier of Corynebacterium glutamicum," J. Biol. Chem., 271(10):5398-5403 (1996) X93514 betP Glycine betaine transport system Peter, H. et al. "Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine," J. Bacteriol., 178(17):5229-5234 (1996) X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of the dapB-ORF2dapA-ORF4 operon ofCorynebacterium glutamicum, encoding two enzymes involved in L-lysine synthesis," Biotechnol. Lett., 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 Corynebacterium glutamicum," Mol.

Microbiol., 22(5):815-826 (1996) 2007203041 29 Jun 2007 Table 2 (continued) X96580 panB; panC; xyIB 3-methyl-2-oxobutanoate Sahm, H. et al. "D-pantothenate synthesis in Corynebacterium glutamicumn and h yd roxyrnethy Itrans ferase; pantoate-beta- use of panBC and genes encoding L-valine synthesis for D-pantothenate alanine ligase; xylulokinase overproduction," App!. Environ. Microbial., 65(5):1973-1979 (1999) X96962 Insertion sequence IS 1207 and transposase X99289 Elongation factor P Ramos, A. et al. "Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofermentumn glutamicumn ATGC 13869)," Gene, 198:217-222 (1997) -OO 14 0 th rB Homoserine kinase Mateos, L.M. et al. "Nucleotide sequence of the homoserine kinase (thrB) gene the Brevibacteriumn lactofernientum," Nucleic Acids Res., 15(9):3922 (1987) Y00151 ddh Meso-diamninopime late D-dehydrogenase Ishino, S. et al. "Nucleotide sequence of the meso-d iam inopi me late D- (EC 1.4.1.16) dehydrogenase gene from Corynebacterium glutamicum," Nucleic Acids Res., (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L.M. et "Nucleotidle sequence of the homoserine dehydrogenase (thrA) gene of the Brevibacterium Iactoferrnentum," Nucleic Acids Res., (1987) Y00546 horn; thrB Homoserine dehydrogenase; homoserine Peoples, O.P. et al. "Nucleotide sequence and fine structural analysis of the kinase Corynebacterium glutam icumn hom-thrB operon," Mo!. Microbiol., 2(1 ):63-72 Y08964 murC; ftsQ/divD; ftsZ UPD-N-acetylmuramate-alanine ligase; Honrubia, M.P. et al. "Identification, characterization, and chromosomal division initiation protein or cell division organization of the ftsZ gene from Brevibacteriumn lactofermentum," Mo!. Gen.

protein; cell division protein Gene., 259(1):97-104 (1998) Y09 163 putP High affinity proline transport system Peter, H. et al. "Isolation of the putP gene of Corynebacterium glutamicumproline and characterization of a low-affinity uptake system for compatible solutes," Arch. Microbiol., 168(2): 143-151 (1997) Y09548 pyc Pyruvate carboxylase Peters- Wend isch, P.G. et "Pyruvate carboxylase from Corynebacterium glutam icum: characterization, expression and inactivation of the pyc gene," Microbiolog, 144:915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et al. "Analysis of the leuB gene from Corynebacterium glutam icum," App!. Microbial. Biotechnol., 50(1 ):42-47 (1998) Y 12472 Attachment site bacteriophage Phi- 16 Moreau, S. et "Site-specific integration of coryncphage Phi- 16: The of an integration vector," Microbial., 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, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine carrier, EctP,"J Bacterial., 180(22):6005-6012 (1998) 2007203041 29 Jun 2007 able 2 (confinqued) Y 1322 1 gInA -Glutarnine s5nthetase I Jakoby, M. et "Isolation of Corynebacterium glutamicum gInA gene .encoding glutamn inc synthetase FEMS MicrabioL Left., 154(1 1-88 (1997) Y 16642 lpd Dihydrolipoamide dehydrogenase Y 18059 Attachment site Corynephage 304L Moreau, S. et al. "Analysis of the integration functions of φ304L: An integrase module among corynephages," Virology, 255(l):150-159 (1999) -Z2150 argS; lysA -Arginyl-tRNA synthetase; diaminopimelate Oguiza, J.A. et al. "A gene encoding arginyl-tRNA synthietase is located in thedecarboxylase (partial) upstream region of the lysA gene in Brevibacterium lactofermentum: Regulation of argS-lysA cluster expression by arginine," J.

Bacleriol., I175(22):7356-7362 (1993) Z2 1502 dapA; dapB Dihydrodipicolinate synthase; Pisabarro, A. et al. "A cluster of three genes (dapA, orf2, and -dapB) of dihydrodipicolinate reductase Brevibacterium lactofermentum encodes dihydrodipicolinate reductase, and a third polypeptide of unknown function," J Bacterial, 1 75(9):2743-2749 (1993) Z29563 thrC Threonine 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 Baclerial, 178(2):550- (1996) Z49823 galE; dtxR Catalytic activity UDP-galactose 4- Oguiza, J.A. et al "The galE gene encoding the UDP-galactose 4-epimerase of epimerase; diphtheria toxin regulatory Brevibacterium lactofennentum is coupled transcriptionally to the dmdR gene," Gene, 177:103-107 (1996) Z49824 orflI; sigB SigB sigma factor Oguiza, J.A. et al "Multiple sigma factor genes in Brevibacterium lactofermnentum: Characterization of sigA and sigB," J Bacterial, 178(2):550- 553 (1996) Z66534 Transposase Correia, A. et al. "Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentumi ATCC 13869," Gene, 70(1):91-94 (1996) A sequence for this gene was published in the indicated reference. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region: TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention Brviaceru amoiges 21054 QTQ- Brevibacterium ammoniagenes 219350 Brevibacterium ammoniagenes 19351 Brevibacteriuin ammoniagenes 19352 Brevibacterium ammoniagenes .19353 Brevibacterium ammoniagenes 193543___ Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenies 10355 Brevibacterium ammoniagenes 2107755__ Brevibacterium ammoniageries 21073 Brevibacterium ammoniagenes 21550 Brevibacterium ammoniagenes 39150 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brcvibacteriurn flavum 21129 Brcvibacterium flavum 21518 Brcvibacterium flavum BI 11474 Brevibacterium flavum BI 11472 Brevibacterium flavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brcvibactcrium flavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B] 11477 Brevibacterium flavum 11478 Brevibacterium flavum 21127 Brevibacterium fiavum BI 11474 Brevi,6acterium healii 15527 Brevibacterium ketoglutamicum 21004 Breviojacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevi-bacterium lactofer-mentum Brevibacterium lacrofermenrum 74 Brevibacterium lactofermentum 77 Brevibacteriuni Iactoferrnentum 21798 Brevibacterium lactofermentum 21799 Brevibacterium Iacroferrnentum 21800 Brevibacteriurn lactoferrnentum 21801 Brevibacterium lactofermentum B] 11470 Brevibacterium lactofermenrum B] 11471 87 G enus- spJej. ATC C FER-M*-NRRt. CECT,- NCGIMB CBS NC DSM1Z IBrevibacterium lactofermentumn 2 1086 Brevibacterium lactofermentumn 21420 Brevibacterium lactofermentum 21086 Brevibacterium lactofer-mentum 31269 Brevibacterium linens 9174 Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacterium paraffinolyticumn 11 160 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 Ccrynebacterium acetoacidophilum 21476 Corynebacterium acetoacidophilum 13870 Ccrynebacterium acetoglutamnicumn B 11473 Ccrynebacterium acetoglutamicumn B 11475 Ccrynebacterium acetoglutamnicumn 15806 Corynebacterium acetoglutamicum 21491 Ccrynebacterium acetoglutrmicumn 3 1270 Ccrynebacterium acetophilum 83671 Corynebacterium arnmoniagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacterium glutrmicumn 14067 Corynebacterium giutrmicumn 39137 Corynebacterium glutamnicumn 21254 Corynebacterium glutamnicumn 21255 Corynebacterium glutrmicumn 3 1830 Corynebacterium glutrmicumn 13032 Corynebacterium glutamicumn 14305 Cory nebacterium glutrmicumn 15455 Corynebacterium glutamnicumn 13058 Cor-ynebacteriurn glutamnicumn 13059 Corynebaccerium glutamicum 13060 Corynebacterium glutamicum 21492 Corynebacterium glutamnicumn 21513 Corynebacterium glutarnicum 21526 Corynebacterium glutamnicumn 21543 Corynebacteriumn glutainicum 13287 Corynebacterium glutrmicumn 21851 Corynebacterium glutamnicumn 21253 Corynebacterium glutamnicumn 21514 Corynebacterium glutamnicumn 21516 Corynebacterium glutamicum 21299

;Z

CA

88 CECT- G C Bs -:NT Q IDSM-Z- Cor-ynebacterium glutarnicum 21300 Gorynebacterium glutamicum 39684 Corynebacterium glutarnicum 21488 Corynebacterium glutarnicum 21649 Corynebacterium glutaxnicum 21650 Corynebacterium glutamicurn 19223 Corynebacterium glutamicum 13869 Corynebacterium glutamicum 21157 Corynebacterium glucamicum 21158 Corynebacterium glutamicum 21159 Corynebacrerium glutamicum 21355 Corynebacierium glutarnicum 31808 Corynebacterium glutamicum 21674 Corynebacterium glutamicum 21562 Corynebacterium glutarnicum 21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum 21565 Co-ynebacterium glutamicum 21566 Cor-ynebacterium glutamicum 21567 Corynebacterium glutamicum 21568 Coryriebacterium glutamicum 21569 Corynebacterium glutamicum 21570 Coryiebacterium glutamicum 21571 Cory.1e bacterium glutam icum 21572 Coryctebacterium glutarnicum 21573 Coryiiebacterium glutamicum 21579 Coryiebacterium glutamicum 19049 Coryiebacterium glutamicum 19050 Cory:nebacterium glutamicum 19051 Coryinebactcrium glutamicum 19052 Corynebacterium glutarnicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 (~~ebaterum glutamicum 19059 Gory ebacterium glutamicum 19060 Corynebacterium glutamicum 19185 Corynebacterium giutamicum 13286 Coryncbacterium glutamnicum 21515 Corynebacterium glutamicum 21527 Corynebacteriurn glutamicurn .21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicurn B8183 Corynebacterium glutamicum Corynebacterium giutarnicum ___B124161 ,Coryylebacterium glutamicum B2l 89 Corynebacterium glutamicum B 12418 Corynebacterium glutamicum B] 11476 Corynebacterium glutamicum 21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium Spec. P4445 Corynebacterium Spec. P4446 Corynebacterium Spec. 31088 Corynebacterium Spec. 31089 Corynebacterium Spec. 3 1090 Corynebacterium Spec. 31090 Corynebacterium Spec. 31090 Corynebacterium Spec. 15954 20145 Corynebacterium Spec. 21857 Corynebacterium Spec. 21862 Corynebacterium Spec. 21863 ATCC: American Type Culture Collection, Rockville, MD, USA FERM: Fermentation Research Institute, Chiba, Japan NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor Schimmelcultures, Baam, NL NCTC: National Collection of Type Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germnany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fuingi and yeasts 4 1h edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.

2007203041 29 Jun 2007 Table 4: Alignment Results Length Accession Name of Genbank Hlt ID length Genbank Hit

(NT)

Source of Genbank Hit homology Date of (GAP) Deposit 37.148 13-Jul-99 rxaOO0l3 996 GB-GSS4:A0713475 581 A0713475 GBHTG3:AC007420 130583 AC007420 GBHTG3:AC007420 130583 AC007420 rxaOO014 903 GBBA1:MTCY3A2 25830 Z83867 HS_5402_B2_Al2_T7A RPCI-1 1 Human Male BAC Library Honmo sapiens Homo sapiens genomic clone Plate=978 Col=24 Row=B, genomic survey sequence.

Drosophila melanogaster chromosome 2 clone BACIRO7M1O (0630) RPCI-98 Drosophila melanogaster 07.M.10 map 24A-24D strain y; cn bw sp, -SEQUENCING IN PROGRESS '.83 unordered pieces.

Drosophila melanogasler chromosome 2 clone BACR07M1O (0630) RPCI-98 Drosophila melanogaster 07.M.10 map 24A-24D strain y: cn bw sp, SEQUENCING IN PROGRESS-% 83 unordered pieces.

34.568 34,568 20-Sep-99 20-Sep-99 GBBA1:MLCB1779 GBBA1:SAPURCLUS rxaOOO3O 513 GBEST21:089713 GBEST28:A1497294 GB_EST2I:C92167 nxa00032 1632 GBBA2:AF010496 43254 9120 767 Z98271 X92429 C89713 Mycobacterium, tuberculosis H37Rv complete genome; segment 136/162. Mycobadterium 58,140 tuberculosis Mycobaclerium leprae cosmid B 1779. Mycobacterium ieprae 57,589 Salboniger napH, pur7, pur'1O, pur6, pur4., pur5 and pur3 genes. Streptomyces anulatus 55.667 089713 Dictyostelium discoideum SS (H.Urushihara) Dictyoslelium discoideum Dictyostelium discoideum 45,283 cIDNA clone SSG229, mRNA sequence.

1b63g03.yi Zebralish WashU MPIMG EST Danio rerio cDNA 5' similar to Danio rerlo 42,991 SW:AFP4_MYQOC P80961 ANTIFREEZE PROTEIN 15-12. mRNA sequence.

C92 167 Dictyosteliumn discoideum SS (H.Urushihara) Dictyosleliumn discoideumn Dictyosteliumn discoideumn 44,444 cDNA clone SSID179, mRNA sequence.

Rhodobactor capsulatus strain SB1003, partial genome. Rhodobacter capsulalus 39,689 8-Aug-97 28-Feb-96 20-Apr-98 17-Jun-98 484 A1497294 637 C92167 189370 AF010496 11 -MAR-1 999 12-Jul-99 GBBA2:AF018073 GBBA2:AF045245 EM-PAT:E1 1760 GBPAT:126124 GB_1N1:LMFL5883 9810 AF018073 Rhodobacter sphaeroides operon regulator (smoC), periplasmic sorbitol-binding Rhodobacter sphaeroides 48,045 protein (smoE), sorbftotlmannitol transport inner membrane protein (smoF), sorbitol/mannitol transport inner membrane protein (smoG). so,'bitot/mannitoi transport ATP-blnding transport protein (smoK), sorbitol dehydrogenase (smoS), mannitol dehydrogenase (mIK), and periplasmic mannitol-binding protein (smoM) genes, complete cds.

5930 AF045245 Kiebsiella pneumoniae D-arabinitol transporter (dalT). D-arabinitot kInase Kiebsiella pneumonlae 38,514 (daIK), 0-arabinitol dehydrogenase (datO)), and repressor (daiR) genes, complete cds.

6911 E11760 Base sequence of sucrase gene. Corynebacterium 99,031 12-MAY-I 996 22-OCT-i 997 16-Jul-98 rxa00041 1342 08-OCT-1 997 glutamicumn 6911 126124 Sequence 4 from patent US 5556776. Unknown.

31934 ALl 17384 Leishmania major Friedlin chromosome 23 cosmid L5883, complete sequence. Leishmania major (Rel. 52, Created) 99,031 07-OCT-I 996 43,663 21-0CT-1999 94,767 08-OCT-i1991 (Rel. 52, Created) 94,767 07-OCT-1996 rxaOO042 882 EM_PAT:E1 1760 GBPAT:126124 6911 E 11760 Base sequence of sucrase gene.

6911 126124 Sequence 4 from patent US 5556776.

Corynebacterium glutamicum Unknown.

2007203041 29 Jun 2007 "a00043 1287 GOIN1:CEU33051 GB PAT: 126124 EMPAT:E1 1760 4899 U33051 6911 126124 6911 El11760 Table 4 (continued) Caenorhabditis elegans sur-2 mRNA. complete cds.

Sequence 4 from patent US 5556776.

Base sequence of sucrase gene.

Homo sapiens clone UWGC:g1564a012 from 7 p'14-15, complete sequence.

Mycobacterium smegmalis phosphogiucose isomerase gene, complete cds.

Caenorhabditis elegans Unknown.

Corynebacterium glutamicum 40,276 97.591 97.591 GB_-PR3:AC005174 39769 rxaOO098 1743 OB-BAl :MSU88433 1928 GBBA1:SC5A7 40337 GB_8A1:MTCY1OD7 39800 r~a001 48 2334 GB-BA1 :MT0Y277 38300 GBBA1:MSGY456 37316 GBBA1:MSGY175 18106 rxa00149 1971 GSBBA1:MSGY456 37316 GBBA1:MSGYI75 18106 GBBA1:MTCY277 38300 684 GBBA1:MTCY274 39991 GB-BAI:MSGB1529CS 36985 GBBA1:MTCY274 39991 rxaOOl96 738 GBBA1:MTCY274 39991 GBBAI:MTCY274 39991 GORO:RATCBRQ 10752 rxa00202 1065 GB-ESTI I:AA253618 313 GB-EST26:A1390284 490 AC0051 74 U88433 AL031 1107 Z79700 Z79701 AD00O001 AD000015 ADOOCOOlI ADOG001 5 Z79701 Z74024 L78824 Z74 024 Z74 024 Z74024 M55532 AA25361 8 A1390284 A1390280 Z99263 AL02 1287 Homo sapiens 35,879 Mycobacterium smegmatls 62,658 Streptomyces coelicolor cosmid 5A7. Streptomyces coelicolor Mycobaclerlum tuberculosis H37Rv complete genome: segment 44/162. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 65/162. Mycobacterium tuberculosis Mycobacterlumn tuberculosis sequence from clone y 4 56. Mycobacterium tuberculosis Myc-obacterium tuberculosis sequence from clone yl 75. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone y456. Mycobacterium tuberculosis Mycobacterium tuberculosis sequence from clone y 1 75. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 65/162. Mycobacterium tuberculosis Mycobacterium tuberculosis H-37Rv complete genome; segment 126/162. Mycobacterium tuberculosis Mycobacterium leprae cosmid B1529 DNA sequence. Mycobacterium leprae Mycobacterium tuberculosis H37Rv complete genome; segment 126/162. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 126/1 62. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 126/162. Mycobacterium tuberculosis Rat carbohydrate binding receptor gene, complete cds. Rattus norvegicus mw9SclO-rl Soares mouse NMVL Mus musculus cONA clone IMAGE:678450 Mus musculus mRNA sequence.

mw96a03.yl Soares mouse NML Mus musculus cONA clone IMAGE:678508 5'Mus musculus similar to TR:0091171 009171 BETAINE-HOMOCYSTEINE METHYLTRANSFERASE;, mRNA sequence.

mw95clO.yl Scares mouse NMVL Mus musculus cDNA clone IMAGE:678450 Mus musculus mRNA sequence.

Mycobacterium leprae cosmid 6637. Mycobacterium leprae Mycobacterium tuberculosis H37Rv complete genome; segment 132/1 62. Mycobacterium tuberculosis 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 3B,816 42,239 37,307 58,312 36,632 23-JanT.96 08-OCT-1997 (Rel. 52, Created) 24-Jun-98 19-Apr-97 27-Jul-98 1 7-Jun-98 17-Jun-98 03-DEC-1 996 1 0-DEC- 1996 03-DEC-i1996 10-DEC-1996 17-Jun-98 19-Jun-98 1 5-Jun-96 19-Jun-98 19-Jun-98 19-Jun-98 27-Apr-93 13-MAR-i1997 2-Feb-99 GB-EST26:A1390280 GBOBAl :MLCB637 GB-BAI:MTV012 467 44882 70287 2-Feb-99 17-Sep-97 23-Jun-99 rxaOO206 1161 2007203041 29 Jun 2007 (GR RA1:-C.6E10 rxa00224 1074 GBBA1:BJU32230 GBBA1 :PDEETFAB GBHTG3:AC009689 rxaD0225 909 GBRO:AF060178 GBGSS11:A0325043 GBEST31:A1676413 rxa00235 1398 GB-BA1:MTCY1002 GB-BA2:AFO61 753 GBBA2:AF086791 rxa00246 1158 GBBA2:AF012550 GB-PAT:E03856 GB_BA1:BACADHT rxa00251 831 GB-BA1:MTCY20G9 GBBA1:MTVOO4 GBBA1:MTVOO4 rxa00288 1134 GB_8A2:AF050114 GBGSS3:B16984 GB IN2:AFi44549 rxa00293 1035 GBEST1:T28483 23990 1769 2440 177954 2057 734 551 38970 3721 37867 2690 1506 1688 37218 69350 69350 1038 469 7887 313 ALl 09661 U32230 L14864 AC009689 AF060 178 A0325043 A1676413 Z92539 AF061 753 AF086791 AF012550 E03856 D90421 Z77 162 AL0091 98 AL009 198 AF0501 14 816984 AF 144549 T28483 Table 4 (continued) Streptomryces Coelicolor cosmrid 6E 10. Streptomyres coelicolo.r 38,616 A3(2) Bradyrhizobium japonicum electron transfer tiavoprotein small subunit (etfS) nd Bradyrhizobium Japonicum 48,038 large subunit (etfL) genes, complete cds.

Paracoccus denitrific-ans electron transfer flavoprotein alpha and beta subunit Paracoccus denitriticans 48,351 genes, complete cdlss.

Homo sapiens chromosome 4 clone 104F_7 map 4, LOW-PASS SEQUENCE Homo sapiens 38,756

SAMPLING.

Mus muscutus heparan sulfate 2-suifotransferase (H-s2st) mRNA, complete cds.Mus musculus 39,506 mgxbOO20J0l r CUGI Rice Blast BAG Library Magnaporthe grisea genomic Magnaporthe grisea 38,333 ctone mgxbOO20JOilr, genoniic survey sequence.

etmEST0l67 EIHi Elmefia teneita cDNA clone etmc074 mRNA sequence. Elmeria tenelta 35,542 Mycobacterium tuberculosis H37Rv complete genome; segment 47/162. Mycobacterlum 65.759 tuberculosis Nitrosomonas europaea CTP synlhase (pyrG) gene, partial cdls; and enolase Nitrosomonas europaea 58,941 (eno) gene, complete cds.

Zymomonas mobilis strain ZM4 clone 67E 10 carbamnoyiphosphate synthetase Zymomonas mohilis 61,239 small subunit (carA), carbamoylphosphate synthetase large subunit (carB), transcription elongation factor (greA), enolase (eno), pyruvate dehydrogenase alpha subunit (pdhA). pyruvate dlehydrogenase beta subunit (pdhB).

ribonuclease H (rnh), homoserine klnase homolog. alcohol dlehydrogenase 11 (adhB), and excinuclease ABC subunit A (uvrA) genes, complete cdls; and unknown genes.

Acinetobacter sp. 80413 ComP (comP) gene, complete cds. Acinetobacter sp. B0413 53,726 gDNA encoding alcohol dlehydrogenase. Bacillus 51 .688 stearothermophlus B.stearothermophilus adhT gene for alcohol dehydrogenase. Bacillus 51,602 stearotherm~ophilus Mycobacteriumn tuberculosis H37Rv complete genome; segment 25/162. Mycobacteriumn 42,875 tuberculosis Mycobacterium tuberculosis H-37Rv complete genome; segment 144/1 62. Mycobacterium 40,380 tuberculosIs Mycobacteriumn tuberculosis H37Rv complete genome; segment 144/1 62. Mycobacterium 41,789 tuberculosis Pseudomonas sp. W 7 alginate lyase gene, complete cds. Pseudomonas sp. W7 49,898 344A1 4.TVC ClT978SKA1 Homo sapiens genomic dlone A-344A1 4, genomic H-omo sapiens 39,355 survey sequence.

Aedes albopictus ribosomal protein L34 (rpi34) gene, complete cds. Aedes atboplctus 36,509 EST46182 Human Kidney Homo sapiens CONA 3' end similar to flavin- Homo sapiens 42,997 containing monooxygenase 1 (HT:1 956), mRNA sequence.

25-MAY-1996 27-OCT-1993 28-Aug-99 18-Jun-98 8-Jan-99 1 9-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-95 3-Jun-99 6-Sep-95 2007203041 29 Jun 2007 GB_-PRI:HUMFMO1 2134 M64082 GBEST32:A1734238 512 A1734238 rxa00296 2967 GBHTG6:ACOI 1069 168266 AC01 1069 GB-EST15AA53168 GB_HTG6:ACO1 1069 rxaOO310 558 GBVI:VMVY1678O GBVi:VARCG GBVi:WVCGAA rxaOO317 777 GBHTG3:AC009571 GBHTG3:ACOODS7I GBPR3:AC005697 rxa00327 507 GB_BAl :LCATPASEB GB_BA1:LCATPASEB rxa00328 615 GBBAi:STYPUTPE GBBA1:ST-YPUTPF

GB_BAI:STYPUTPI

rxa00329 1347 GBPR3:AC004691 GB PR4AC004916 GBPR3:AC004691 rxaOO340 1269 GBBA1:MTCY427 GB-GSS1 2:AQ4 12290 GBPL2:AF112871 rxa00379 307 GB-HTG1:CEY56A3 GBHTG1:CEY56A3 414 168266 AA53 1468 AC01 1069 186986 Y16780 186103 L22579 185578 X69198 159648 AC009571 159648 AC009571 174503 AC005697 1514 X64542 1514 X64542 1887 L01138 1887 L01139 1889 L01 142 141990 AC004691 129014 AC004916 141990 AC004691 38110 Z70692 238 AQ412290 2394 AF112871 224746 AL022280 224746 AL022280 Table_4 (continue-d1 Human flavin-containing monooxygenase (FMOi) mRNA, complete cds.

zb73c05.y5 Soares fetal-lungNbHL1 9W Homo sapiens cONA clone IMAGE:309224 5' similar to gb:M64082 DIMETHYLANILINE MONOOXYGENASE (HUMAN);, mRNA sequence.

Drosophila melanogaster chromosome X clone BACR1 1H20 (0881) RPCI-98 1 1.H.20 map 12B-12C strain y; cn bw sp, SEQUENCING IN PROGRESS -,92 unordered pieces.

nj63d1 2.sI1 NCICGAP_Prl10 Homo sapiens cDNA clone IMAGE:997175, mRNA sequence.

Drosophila melanogaster chromosome X clone BACRI I H20 (0881) RPCI-98 1l1.H.20 map 12B-12C strain y; cn bw sp, -SEQUENCING IN PROGRESS 92 unordered pieces.

variola minor virus complete genome.

Varlola 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 '1 clone 57_A_22 map 4, -SEQUENCING IN PROGRESS 8 unordered pieces.

Homo sapiens chromosome 17, clone hRPK.138-P_22, complete sequence.

casei gene for ATPase beta-subunit.

L.casei gene for ATPase beta-subunit.

Salmonella (S2980) proline permease (putP) gene, 5' end.

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

Salmonella (S3015) proline permease (pulP) gene, 5' end.

Homo sapiens PAC clone 0J10740002 from 7p14-p1 5, complete sequence.

Homo sapiens clone D.106911-14, complete sequence.

Homo sapiens PAC clone 0J10740002 from 7p14-p15, complete sequence.

Mycobacterium tuberculosis H37Rv complete genome: segment 99/162.

RPCI-1 1-195H2.TV RPCI-1 1 Homo sapiens genomic clone RPCI-1 1-1 95H2, genomic survey sequence.

Aslasia longa small subunit ribosomal RNA gene, complete sequence.

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

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

Homo sapiens Homo sapiens 37,915 8-Nov-94 41,502 14-Jun-99 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 Astasia longa Caenorhabditis elegans Caenorhabdilis elegans 40,82f 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 38,940 36,555 36,465 35,.179 35.179 Drosophila melanogasler 33,890 02-DEC-i1999 20-Aug.97 02-DEC-I1999 2-Sep-99 12-Jan-95 1 3-DEC-1996 29-Sep-99 29-Sep-99 09-OCT-i1998 I11-DEC-i 992 1 1-DEC-1992 09-MAY-I1996 09-MAY-i1996 09-MAY- 1996 16-MAY-I1998 17-Jul-99 16-MAY-I1998 24-Jun-99 23-MAR-i1999 28-Jun-99 6-Sep-99 6-Sep-99 2007203041 29 Jun 2007 G8-PR2:HS134019 GBGSS4:AQ730532 86897 416 rxaOO381 729 Table 4 (continued) AL034555 Human DNA sequence from clone 134019 on chromosome I1p36.11-36.33, Homo sapiens complete sequence.

AQ730532 HS_2149_Al_C06_T7WC CIT Approved Human Genomic Sperm Library D Homo sapiens Homo sapiens genomic clone Plate=2149 ColI'1 1 Royr-E, genomic survey sequence.

At 120939 ub74f05.ri Soares mouse mammary gland NMLMG Mus musculus cDNA clone Mus musculus IMAGE:11383489 5 similar to gb:J04046 CALMODULIN (HUMAN): gb:M19381 Mouse calmodulin (MOUSE);, mRNA sequence.

A1120939 ub74fO5.rl Soares mouse mammary gland NMLMG Mus musculus cONA clone Mus musculus 40,604 35,766 23-Nov-99 1 5-Jul-99 GBE5T23:AI120939 561 GBEST23:A1120939 561 41,113 2-Sep-98 41.113 2-Sep-98 rxa00385 362 rxaOO388 1134 rxa00427 909 rxa00483 1587 rxaOO511 615 GBE5T32:A1726450 GBG554:A0740856 GB-PRi:HSPAIP GB_BAi:MTY25DIO GB-BA1:MSGY224 GBHTG1:AP000471 GBBA1:MSGYi26 GBBAI:MTYi3D12 GBHTGI:CEY48C3 GBPR2:HSAFOOI55O GBBAI:LLCPJW565 GBHTG2:AC006754 GB_PR3:HSE127C11 565 768 1587 40838 40051 72466 37164 37085 270193 173882 12828 206217 38423 38423 22550 A1726450 AQ740856 X91809 Z95558 AD000004 AP000471 AD000012 Z80343 Z92855 AFO0l 550 Y12736 AC006754 Z74581 Z74581 Z95585 IMAGE:1383489 5' similar to gb:J04046 CALMODULIN (HUMAN); gb:M19381 Mouse calmodulin (MOUSE);, mRNA sequence.

BNLGHi5857 Six-day Cotton fiber Gossypium hirsutum cDNA 5' similar to (AF0l 5913) Skbi1Hs [Homo sapiens]. mRNA sequence.

HS_2274_A2_A07_T7C CIT Approved Human Genomic Sperm Library 0 Homo sapiens geriomic clone Plate=2274 Col=14 Row--A, genomic survey sequence.

H.sapiens mRNA for GAIP protein.

Mycobacterium tuberculosis H37Rv complete genome: segment 28/162.

Mycobacterium tuberculosis sequence from clone y224.

Homo sapiens chromosome 21 clone B2308H 15 map 21q22.3.

SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterium tuberculosis sequence from clone y126.

Mycobacterium tuberculosis H37Rv complete genome; segment 156/162.

Caenorhabditis elegans chromosome 11 clone Y48C3, SEQUENCING IN PROGRESS In unordered pieces.

Homo sapiens chromosome 16 BAC clone CIT987SK-334D 11 complete sequence.

Lactococcus lactis cremoris plasmid pJW565 DNA, abiiM, abiiR genes and o rfX.

Caenorhabditis elegans clone Y4OBlO, -SEQUENCING IN PROGRESS unordered pieces.

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

Human DNA sequence from cosmid El127C1 1 on chromosome 22q1 1.2-qter contains STS.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.

Gossypium hirsutum Homo sapiens Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis Caenorhabditis elegans Homo sapiens Lactococcus lactis subsp.

cremoris Caenorhabditis elegans Homo sapiens Homo sapiens Mycobacterium tuberculosis 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 1 1-Jun-99 16-Jul-99 29-MAR-i1996 17-Jun-98 03-DEC-1996 10-DEC-i1996 17-Jun-98 29-MAY-1999 22-Aug-97 01-MAR- 1999 23-Feb-99 23-Nov-99 GBPR3:HSEi27CI1 rxaOO5I2 718 GB-BA1:MTCY22G8 23-Nov-99 17-Jun-98 2007203041 29 Jun 2007 Table 4 (continued) M.smegmatis gltA gene for citrate synthase.

GBBA1:MSGLTA 1776 X60513 Mycobacterium smegmatis 56,143 20-Sep-91 GBBA2:ECU73857 ra00517 1164 GBHTG2:AC006911 GBHTG2:AC00691 1 GB-EST29:A1602 158 rxa00518 320 GBBA2:ECU73857 GBBA2:STU51879 rxaOO606 2378 rxa00635 1860 GBBA2:AE000140 GBEST32:AU068253 GBEST13:AA363046 GBEST32:AU068253 GBBA1:PAORFI GBBA1:PAORF1 128824 298804 298804 481 128824 8371 12498 376 329 376 1440 11440 80381 81493 80381 38400 81493 43481 197110 197110 181745 U73857 AC00691 1 AC00691 1 A1602 158 U73857 U5 1879 AE000 140 AU068253 AA363046 AU068253 Xi 13378 X13378 AC01 0871 X98130 AC010871 AC004058 X98 130 AB026648 AC01 0325 AC01 0325 AC0081 79 Escherichia coi chromosome minutes 6-8.

Caenorhabditls elegans clone Y94H-6x, -SEQUENCING IN PROGRESS ,Caenorhabditis elegans unordered pieces.

Caenorhabditis elegans clone Y941-16x, -SEQUENCING IN PROGRESS Caenorhabditis etegans unordered pieces.

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

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

Escherichia coll K-12 MG1655 section 30 of 400 of the complete genome. Escherlchia coti AU068253 Rice callus Oryza saliva cDNA clone C12658_9A, mRNA sequence. Oryza saliva EST72922 Ovary 11 Homo sapiens cDNA 5' end, mRNA sequence. Homo sapiens AU068253 Rice callus Oryza saliva cDNA clone C12658_9A, mRNA sequence. Oryza saliva Pseudomonas amyloderamosa DNA for ORF 1. Pseudomonas amyloderamosa Pseudomonas amyloderamosa DNA for ORF 1. Pseudomonas amyloderamosa Arabidopsis thallana chromosome III BAC T16011I genomic sequence, Arabidopsis thaliana complete sequence.

Athaliana 81 kb genomic sequence. Arabidopsis thatiana Arabidopsis thaliana chromosome III BAG T1601 1 genomlc sequence, Arabidopsis thaliana complete sequence.

Homo sapiens chromosome 4 clone B241 P1 9 map 4q25, complete sequence. H-omo sapiens Athaliana 81kb genomic sequence. Arabidopsis; thaliana Arabidopsis thaliana genomic DNA, chromosome 3. PI clone: MLJI5, complete Arabidopsis thaiiana sequence.

Homo sapiens chromosome 19 done CITB-E 12568A1 7, SEQUENCING IN Homo sapiens PROGRESS 40 unordered pieces.

Homo sapiens chromosome 19 clone CITB-E1_2568A1 7, SEQUENCING IN Homo sapiens PROGRESS 40 unordered pieces.

Homo sapiens clone NH-0576F 01. complete sequence. Homo sapiens 37.889 37,889 40,833 49,688 50,313 49,688 41.333 34,347 41,899 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' AR-i 997 13-Nov-99 30-Sep-98 1 2-MAR-I1997 07-MAY-i1999 15-Sep-99 1 5-Sep-99 28-Sep-99 Escherichia coi 48,563 14-Jul-99 rxa00679 1389 GBPL2:AC010871 GBOPL :A181 KBGEN GB-PL2:ACO1 0871 rxaOO68O 441 GBPR3:AC004058 GB P1:AT81 KBGEN GB-P1:AB026648 rxa00682 2022 GBHTG3:AC010325 GB-HTG3:ACO1 0325 GB-PR4:ACOO8I 79 2007203041 29 Jun 2007 rxa00683 1215 GBBA2:AE000896 10707 GBINI:DMBR7A4 212734 GBEST35:AV163010 273 rxa00686 927 GBHTG2:HSDJ137K2 190223 GBHTG2:HSDJ137K2 190223 GBESTI2:AA284399 431 AE000896 ALl109630 AV163010 AL049820 AL049820 AA284 399 A1785570 rxaOO700 927 GBEST34:AI785570 GB-EST25:A1256 147 GBBAI:CARCGI2 rxaOO703 2409 GBBA1:SC7H2 GBBAl :MTCY274 GBBA2:REU60056 rxaOO705 1038 GBGSSi5:A0604477 GBEST1 1AA224340 GBEST5:N30648 rxa00782 1005 GB-BA1:MTCY1007 GBBAI:MLCL373 GBBA2:AF128399 rxa00783 1395 GBHTG2:ACOO815B GBHTG2:ACOO8 158 GBPR3:AC005017 rxa00794 1128 GBBA1:MTV017 Tabie 4 (continued) Mehanohacteriurm fhnrmnritntrnnhiriom fren hnp 1 IcAUQ- i9rnoAc (section 102 of 148) of the complete genome.

Drosophila melanogaster clone BACR7A4.

AV163010 Mus musculus head C57BU/6J 13-day embryo Mus musculus cIDNA clone 3110006J22. mRNA sequence.

Homo sapiens chromosome 6 clone RP1-1I37K2 map q25.1-25.3, SEQUENCING IN PROGRESS In unordered pieces.

Homo sapiens chromosome 6 clone RPI -1 37K2 map q25.1-25.3, SEQUENCING IN PROGRESS in unordered pieces.

zs57b04.rl NCI_-CGAP GCBI Homo sapiens cIDNA clone IMAGE:701551 5% mRNA sequence.

uj44d03.x1 Sugano mouse liver mija Mus musculus cDNA clone IMAGE: 1922789 3' similar 1o gb:Z28407 60S RIBOSOMAL PROTEIN 18 (HUMAN);, mRNA sequence.

u[95e12.xl Sugano mouse liver mlia Mus musculus cDNA clone IMAGE:1890190 3' sImilar to gb:Z28407 60S RIBOSOMAL PROTEIN L8 (HUMAN):, mRNA sequence.

C. auranlacus reaction center genes I and 2.

Streptomyces coelicolor cos mid 7H2.

Mycobacterium tuberculosis H37Rv complete genome; segment 126/162.

thermoautolrophicumn Drosophila melanogaster Mus musculus Homo sapiens Homo sapiens Homo sapiens Mus musculus Mus musculus Chiorotlexus aurantlacus Streptomyces coelicolor A3(2) Mycobaclerium tuberculosis 36.454 4 1.758 38,031 38,031 39,205 41,943 30-Jul-99 8-Jul-99 03-DEC-i1999 03-DEC-i1999 14-Aug-97 2-Jul-99 684 A1256147 2079 X14979 42655 AL109732 39991 Z74024 2520 U60056 505 A0604477 443 AA224340 291 N30648 39800 Z79700 37304 AL035500 28-42 AF128399 118792 AC008158 118792 AC008158 137176 AC005017 40.791 12-Nov-98 Ralstonla eutropha formate dehydrogenase-like protein (cbb~c) gene, complete Raistonia eutropha cds.

HS_2116_B_-G07-MR ClITApproved Human Genomlc Sperm Library D Homo Homo sapiens sapiens genomlc clone Plalo=21 16 CoI=13 Row=N, genomic survey sequence.

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

yw77b02.sl Soaresjlacenta -8to9weeks_-2NbHP~to9W Homo sapiens cDNA Homo sapiens clone IMAGE:258219 mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome: segment 44/162. Mycobacterium tuberculosis Mycobocterium leprae cosmid L373. Mycobacterium Ieprae Pseudomonas aeruginosa succlnyl-CoA synthetase beta subunit (sucC) and Pseudomonas aeruginosa succinyl-CoA synthetase alpha subunit (sucD) genes, complete cds.

Homo sapiens chromosome 17 clone hRPK.42 _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 BAC clone GS214N13 from 7p14-p15, complete sequence. Homo sapiens 37,72 1 56,646 37,369 51,087 39,6 17 35.129 43,986 63,327 62,300 53,698 35J 35 35,135 35,864 23-Apr-91 2-Aug-99 19-Jun-98 16-OCT-i1996 10-Jun-99 1 1-MAR-1998 5-Jan-96 17-Jun-98 27-Aug-99 25-MAR-i1999 28-Jul-99 28-Jul-99 8-Aug-98 67200 AL021897 Mycobacterium tuberculosis H37Rv complete genome; segment 48/1 62. Mycobacterium tuberculosis 40,331 24-Jun-99 2007203041 29 Jun 2007 GB BAI:MLCB1222 GBPR2:H5151B14 34714 AL049491 128942 Z82188 rxa00799 1767 GBPL2:AF016327 616 AF016327 GBHTG2:HSDJ319M7 128208 AL079341 GBHTG2:HSDJ3i1 M7 128208 AL07934 1 Table 4 (continued) Mycobacterium leprae cosmid B 1222.

Human DNA sequence from clone 15IB14 on chromosome 22 Contains SOMATOSTATIN RECEPTOR TYPE 3 (SS3R) gene,pseudogene similar to ribosomal protein L39,RAC2 (RAS-RELATED 03 BOTULINUM TOXIN SUBSTRATE 2 (P21-RAC2)) gene ESTs, STSs, GSSs and CpG Islands.

complete sequence.

Hordeum vulgare Barpermi (permit 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 RP1-319M7 map p2l.1-2I.3, SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterlumn tuberculosis H37Rv complete genome; segment 100/162.

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

S.cerevisiae SFA and ARP genes.

Mycobacterium ieprae Homo sapiens Hordeum vulgare Homo sapiens Homo sapiens Mycobacterium tuberculosis Streptomyces coellcolor 61,170 37,455 27-Aug-99 16-Jun-99 41,311 01-OCT.1997 36,845 30-Nov-99 36,845 30-Nov-99 rxaOO800 1227 GBBAI:MTVO22 GB-BA1:AB019513 GB PL1:SCSFAARP rxa00825 1056 GB_BA1:MTYISC1O GOBAI:MLCB2548 GBBA2:AF169031 13025 AL021925 4417 A801 9513 7008 X68020 63,101 41,312 17-Jun-98 13-Nov-98 29-Nov-94 Saccharomyces cerevisiae 36,288 33050 Z95436 Mycobacteriumn tuberculosis H37Rv complete genome; segment 1541162.

38916 AL023093 1141 AF169031 rxa00871 rxa00872 1077 GB_INI:CEF23H12 GBHTG2:AC007263 GBIITG2:AC007263 rxa00879 2241 GBBA1:MTVO49 GBPL2:C0U236897

GBPLI:CXACTIA

rxa00909 955 GB-t3A2:AF010496 35564 Z74472 167390 AC007263 167390 AC007263 40360 AL022021 Mycobaclerlum leprae cosmid B2548.

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

Caenorhabditis elegans cosmid F23H 12. complete sequence, Homo sapiens chromosome 14 clone BAC 79J20 map 14q31., SEQUENCING IN PROGRESS ordered pieces.

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

Mycobacterium tuberculosis H37Rv complete genome; segment 81/162.

Candida dubliniensis ACTi gene, exons 1-2.

Candida aibicans actl gene for ectin.

Rhodobacter capsulatus strain SB 1003, partial genome.

Mycobacterium tuberculosis Mycobacterium Ieprae Xanthomonas oryzae pv.

oryze Caenorhabditis etegans Homo sapiens Homo sapiens Mycobacterium tuberculosis Candida dubliniensls Candida albicans Rhodobacter capsulatus Sinortiizobium metiloti 39,980 17-Jun-98 39.435 46,232 27-Aug-99 14-Sep-99 34,502 08-OCT-1999 35,714 24-MAY-i1999 35,714 24-MAY-i1999 36,981 19-Jun-98 1827 3206 189370 AJ236897 X16377 AF010496 38,716 36,610 51.586 1-Sep-99 10-Apr-93 12-MAY-1 998 GB_BA1:RMPHA 7888 X93358 RhIzoblum meliloli phs[AB,C.D,E,F,G] genes. 48,367 12-MAR-1999 GBEST16:C23528 rxaOO9l3 2118 GB-HTG2:AC007734 317 023528 188267 AC007734 023528 Japanese flounder spleen Paralichthys olivaceus cDNA clone HB5(2).

mRNA sequence.

Homo sapiens chromosome 18 clone hRPK44_0)1 map 18, SEQUENCING IN PROGRESS 18 unordered pieces.

Paralichthys otivaceus 41,640 28-Sep-99 Homo sapiens 34,457 5-Jun-99 2007203041 29 Jun 2007 GBHTG2:AC007734 GBEST18:AA709478 rxa00945 1095 GBHTG4:AC010351 GB-HTG4:AC010351 GBBAI:MTCYO5A6 188267 406 220710 220710 38631 AC007734 AA709478 AC010351 ACO 1035 1 Z96072 Table 4 (continued) Homo s apie ns chromosome 18A clone h RPKA4Q1 18, SEQUENCING IN PROGRESS 18 unordered pieces.

vv34a05.rl Siratagene mouse heart (#937316) Mus musculus cONA clone IMAGE: 1224272 5% mRNA sequence.

Homo sapiens chromosome 5 clone GITB-Hi_12022136, -SEQUENCING IN PROGRESS 68 unordered pieces.

Homo sapiens chromosome 5 clone ITB-Hi_2022B6, -SEQUENCING IN PROGRESS 68 unordered pieces.

Mycobactorlum tuberculosis H37Rv complete genome; segment 120/162.

rxa00965 rxa00999 1575 GBPAT:E13660 GBBAI:MTCY359 GBBAI:MLCB1788 442 GB-BA1:MTVOO8 GBBA1:MTVOO8 1916 36021 39228 63033 63033 rxa01025 1119 GBBA1:SC7A1 32039 GBBAi:MSGB1723CS 38477 GBBA1:MLCB637 44882 rxa01O48 13,47 GBBA2:AF017444 3067 GB-BA1:BSUBOO13 218470 GBVI:HSV2HG52 154746 rxaOIO4g 1605 GBHTG2:AC002518 131855 GBHTG2:AC00251 8 131855 GBHTG2:AC00251 8 131855 rxaO1O77 1494 GBPR3:H5DJ65305 85237 GBBA1:ECU29579 72221 GBBA1:ECU29579 72221 rxaOlO89 873 GBG558:AQ044021 387 E13660 Z83859 AL008609 AL02 1246 AL02 1245 AL034447 L78825 Z99263 AF0 17444 Z99 116 Z86099 AC002518 AC002518 AC002518 AL0-49743 U29579 U29579 AQ044021 gDNA encoding 6-phosphogluconate dehydrogenase.

Mycobacterium tuberculosis H37Rv complete genome; segment 84/162.

Mycobaclerium Ieprae cosmid 8 1788.

Mycobacterium tuberculosis H37Rv complete genome; segment 108/162.

Mycobacterium tuberculosis H37Rv complete genome; segment 108/1 62.

Streptomyces coelicolor cosmid 7A1.

Mycobacterium leprae cosmid 81 723 DNA sequence.

Mycobacterium leprae cosmid B637.

Sinorhizobium meliloti NADP-dependent malic enzyme (ine) gene, complete cds.

Bacillus subtills complete genome (section 13 of 21): from 2395261 to 2613730.

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

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

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

Homo sapiens chromosome X clone bWXD20, SEQUENCING IN PROGRESS 11 unordered pieces.

Human DNA sequence from clone 653C5 on chromosome 1p21.3-22.3 Contains CA repeat(D15435), SISs and GSSs. complete sequence.

Escheichla coi K-1 2 genome; approximately 611to62 minutes.

Escherichia c-ol K-12 genome; approximately 611to62 minutes.

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

HiOMO sapieris Mus musculus Homo sapiens Homo sapiens Mycobactedum tuberculosis Corynobacterium glulamicum Mycobacterium tuberculosis Mycobacterium leprae Mycobacterium tuberculosis Mycobacterium tuberculosis Streptomyces coelicolor Mycobacterium leprae Mycobacterlum Ieprae Sinorhizobium meliloti Bacllus subilis human herpesvirus 2 Homo sapiens Homo sapiens Homo sapiens Homo sapiens Escrerichia coi Escherchla coi Honmo sapiens 34,457 42.065 36,448 36,448 36.2 18 98,349 38,520 64,355 39.860 39,.120 55,287 56,847 56,676 53,660 37,255 38.081 35,647 35,647 26,180 36,462 41.808 36,130 36.528 24-Jun-98 17-Jun-98 127- Aug-99 17-Jun-98 5-jun-9y 24-DEC-1 997 31-OCT-1999 31-OCT- 1999 17-Jun-98 15-DEC-1998 15-Jun-96 17-Sep-97 2-Nov-97 26-Nov-97 04-DEC-1 998 2-Sep-97 2-Sep-97 2-Sep-97 23-Nov-99 1-Jul-95 1-Jul-95 14-Jul-98 2007203041 29 Jun 2007 Table 4 (continued) A0042907 CIT-IISP-231 8D1 7.TR CIT-HSP Homo sapiens genomic clone 2318017. GBG558:A0042907 GBGSS8:A0044021 rxaOlO93 1554 GBBA:CORPYKI GB_BA1:MTCY01B2 GBBA1:M1U65430 rxa0lO99 948 GBBA2:AF045998 GBBA2:AF051846 GBGSS1:FR0005503 rxa01lIl 541 GBPR3.AC004063 GB_PR3:HS1 178121 GBHTG3:AC009301 rxaOI13O 687 GBHTG3:AC009444 GBHTG3:AC009444 GBIN1:OMC66A1 rxaOll93 1572 GBBA1:CGASO19 EMPAT:E09634 GBBAI:MLU15186 nxa0l 194 495 EMPAT:E09634 GBBA1:CGASO19 GBVI:HEPCRE4B rxa0 1200 392 387 2795 35938 1439 780 738 619 177014 62268 163369 164587 164587 34127 11452 1452 36241 1452 A0044021I L27 126 Z95554 U65430 AF045998 AF05 1846 Z89313 AC004063 AL 109852 AC009301 AC009444 AC009444 AL031 227 X76875 E09634 U1 5186 E09634 genomic survey sequence.

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

Corynebacterium pyruvate kinase gene, complete eds.

Mycobacterium tuberculosis H37Rv complete genome; segment 72/162.

Mycobacterium Intracellulare pyruvate kinase (pykF) gene, complete cds.

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

Corynebactenium glutamicum phosphoribosylformlmnino-5-amlno-1phosphoribosyl-4- Imidazolecarboxamidle Isomerase (hisA) gene, complete cds.

Frubripes 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_03, -SEQUENCING IN PROGRESS 8 unordered piece s.

Drosophila melanogaster cosmid 66A1.

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

Brevibacteriurn fiavumn UncD gene whose gene product is Involved in Mycobacterium Ieprae cosmid L471.

Brevibacterium flavum UncO gene whose gene product is involved in C.glutamicum (ASO 19) ATPase beta-subunit gene.

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

Homo sapiens H-omo sapiens Corynebacterium glutamlcurn Mycobacterium tuberculosis Mycobacterlumn intracellulare Corynebacterium glutamicum Corynebacterium glutamicumn Fugu rubripes Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Drosophila melanogaster Corynebacterlumn glutamicum Corynebaclerlum glutamicumn Mycobacteriumn leprae Corynebacteriumn glulamicumn Corynebacterium g lutamicum Hepatitis C virus 35.969 14-Jul.98 44.545 100,000 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 07-DEC-1 994 17-Jun-98 23-DEC-i1996 19-Feb-98 12-MAR-I1998 01-MAR-1997 1 0-Jul-98 01-DEC-i1999 13-Aug-99 22-Aug-99 22-Aug-99 05-OCT-i1998 27-OCT-I1994 07-OCT-i1997 (Ret. 52, Created) 09-MAR-I1995 07-OCT-i1997 (Rel. 52, Created) 27-OCT-i1994 5-Apr-92 1452 X76875 414 X60570 2007203041 29 Jun 2007 rxa0120i 1764 GBBA1:SLATPSYNA GB-BAI:MTCY373 GB BA 1:M1U15186 rxa01202 1098 GBBA1:SLATPSYNA GBBA1:SLATPSYNA GBBAi:MCSQSSHC rxa0l2O4 933 GBPLi:AP000423 GBHTG6:AC009762 GBHTG6:AC009762 rxa01216 1124 GBBA1:MTCY1002 GBBA2:AF017435

GBBAI:CCRFLBDBA

rxa01225 1563 GBBA2:AF058302 GBHTG3:AC007301 GBHTG3:AC007301 rxa0i227 444 GBBA1:SERFDXA GBBAI:MTV005 GBl BAl :MSGY348 rxa01242 900 GBPR3:AC005697 GB-HTG3:ACO1 0722 GB-HTG3:ACO 10722 8560 35516 36241 8560 8560 5538 154478 164070 164070 38970 4301 4424 25306 165741 Z22606 Z73419 U15186 Z22606 7-22606 Y09978 AP000423 AC0097E2 AC009762 Z92539 AF0 17435 M69228 AF058302 AC007301 Table 4 (continued) Slividans i Protein and ATP synthase genes.

Mycobacterium tuberculosis H37Rv complete genome; segment 57/162.

Mycobacteriumn leprae cosmid 1-471.

Slividans i protein and ATP synthase genes.

S.Iividans i protein and ATP synthase genes.

M.capsulatus ortc, orly. orfz, sqs and shc genes.

Arabidopsis thaliana chioroplast genomic DNA, complete sequence, strain:Cotumbla.

Homo sapiens clone RP1 1-11416, -SEQUENCING IN PROGRESS ~,39 unordered pieces.

Homo sapiens clone RP1 1-114116, -SEQUENCING IN PROGRESS ,39 unordered pieces.

Mycobacterlumn tuberculosis H37Rv complete genome; segment 471162.

Methylobacterium extorquens methanol oxidation genes. glmU-Iike gene, partial cds, and orfl-2, orft.1, orfR genes. complete cds.

Ocrescenlus flagellar gene promoter region.

Streptomyces roseofulvus frenolicin biosynthetic gene duster, complete sequence.

Drosophila melanogasler chromosome 2 clone BACRO4BO09 (D576) RPCI-98 04.13.9 map 43E1 2-44F1 strain y; cn bw sp, -SEQUENCING IN PROGRESS -,150 unordered pieces.

Drosophila melanogasler chromosome 2 clone BACRO4BO09 (D3576) RPCI-98 04.B.9 map 43EI2-44FI strain y; cn bw sp, -SEQUENCING IN PROGRESS 150 unordered pieces.

Saccharopolyspora erythraea ferredoxin (fdxA) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 51/162.

Mycobacteriumn tuberculosis sequence from clone y348.

Homo sapiens chromosome 17, clone hRPK.138P 22, complete sequence.

Homo sapiens clone NH0122L-09, SEQUENCING IN PROGRESS *,2 unordered pieces.

Homo sapiens dlone NH-0122L-09, -SEQUENCING IN PROGRESS *,2 unordered pieces.

5AirpnlnmyreS !i'.idans Mycobacterium tuberculosis Mycobacterium leprae Streptomyces lividans Streplomyces lividans Meihylococcus capsulatus Chioroplast Arabidopsis thaliana Homo sapiens Homo sapiens Mycobacterium tuberculosis Methylobacteuium extorquens Caulobacter crescenlus Streptomyces roseofulvus Drosophila melanogaster Drosophila melanogaster Saccharopolyspora erythraea Mycobacteriumn tuberculosis Mycobacterium tuberculosis Homo sapiens Homo sapiens Homo sapiens C5.269 65,437 39,302 57,087 38,298 37,626 38,395 35,459 36,117 39,064 42.67 1 41,054 36,205 39,922 39,922 64,908 62.838 61,712 35,373 39,863 39.863 01- MAY. 1995 17-Jun.98 09-MAR-i1995 01-MAY-1995 01-MAY-i 995 26-MAY-i1998 15-Sep-99 04-DEC-I 999 04-DEC-i1999 17-Jun-98 10-MAR-i1998 26-Apr-93 2-Jun-98 17-Aug-99 17-Aug-99 13-MAR-i1996 17-Jun-98 10-DEC-1996 09-OCT-1 998 25-Sep-99 25-Sep-99 165741 AC007301 3869 37840 40056 174503 160723 160723 M61 119 AL010185 AD00O020 AC005697 AC01 0722 AC0 10722 2007203041 29 Jun 2007 rxa01243 1083 GBGSS1O:AQ255057 GB IN1:CEKO5D4 GB-IN1:CEKO5D4 rxa01259 981 GBBA1:CGLPD GBHTG4:ACO 10567 GBHTG4:AC010567 rxa01262 1284 GBBA2:AF1 72324 GBBA2:ECU78086 GB-BAI :D90841 rxa0l311 870 GBPR3:AC004103 GBHTG3:AC007383 GB-HTG3:AC007383 rxa013i2 2142 GBBA2:AE000487 GB BA1:MTVO16 GB SAl :U00022 rxao 1325 795 GBHTG4:AC009245 GBHTG4:A0009245 GBHTG4:AC009245 rxa01332 576 GB-HTG6:AC007186 583 19000 19000 1800 143287 143287 14263 AQ255057 Z92804 Z92804 Y 16642 AC0 10567 AC01 0567 AF172324 Table 4 (continued) mgxbOOO8N0lr CUGl Rice Blast BAG Library Magnaporthe grisea genomic Magnaporthe grisea clone mgxbOOOBN l r, genomic survey sequence.

Caenarhabditis elegans cosmid K05D4, complete sequence. Caenorhabditis elegans Caenorhabditis elegans cosmld K05D4, complete sequence. Caenorhabdllis elogans Corynebacterium glutamicum lpd gene, complete CDS. Corynebacteriumn glutamicumn Drosophila melanogaster chromosome 31J69C1 clone RPCI98-1 1N6. Drosophila melanogasler SEQUENCING IN PROGRESS 70 unordered pieces.

Drosophila melanogaster chromosome 3lJ69C1 clone RPCI98-1 I N6, Drosophila melanogaster -SEQUENCING IN PROGRESS 70 unordered pieces.

Escherichla coil GaIF (gaIF) gene, partial cds; 0-antigen repeat unit transporter Escherichia coil Wzx (wzx). WbnA (wbnA), 0-antigen polymerase Wzy (wzy), WbnB (wbnB).

WbnC (wbnC), WbnD (wbnD), WbnE (wbnE), UDP-Glc-4-epimerase GalE (galE). 6-phosphogluconate dehydrogenase Gnd (gnd), UDP-GIc-6dehydrogenase Ugd (ugd), and WbnF (wbnF) genes, complete cds; and chain length determinant Wzz (wzz) gene, partial cds.

Escherlchia coil hypothetical urldine-5'-dlphosphoglucose dehydrogenase (ugd) Escherlchla coil 38,722 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-OCT-1 999 16-OCT-1 999 29-OCT-i1999 4759 U78086 20226 D90841 144368 AC0O4103 215529 AC007383 215529 AC007383 13889 AE000487 53662 AL021841 36411 U00022 215767 AC009245 215767 AC009245 215767 AC009245 225851 AC007186 59.735 5-Nov-97 and 0-chaIn length regulator (wzz) genes, complete cds.

E.coli genomic; DNA, Kohara clone #351(45.1-45.5 min.).

Homo sapiens Xp22 SAC GS-6119J13 (Genome Systems Human BAG library) complete sequence, H-omo sapiens clone NHO131O11, -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.

Mycobacteriumn tuberculosis H37Rv complete genome; segment 143/1 62.

Mycobacterlumn leprae cosmid L308.

Homo sapiens chromosome SEQUENCING IN PROGRESS 24 unordered pieces.

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

Homo sapiens chromosome 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; cn bw sp, -SEQUENCING IN PROGRESS unordered pieces.

Escherichia coli Homo sapiens Homo sapiens Homo sapiens Escherichla coi Mycobacterum tuberculosis Mycobacterium Ieprae Homo sapiens Homo sapiens Homo sapiens Drosophila melanogasler 37,904 37,340 36,385 36,385 39,494 46,252 46,368 36,016 36,016 39,618 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-I1999 GBHTG6:AC007147 202291 AC007147 Drosophila melanogaster 35,366 2007203041 29 Jun 2007 Table 4 (continued) Homo sapiens clone RPCI 11-375120, -**SEQUENCING IN PROGRESS-,. 25 Homo sapiens 1107 rxa0i365 1497 rxa01369 1305 rxa01377 1209 rxa01392 1200 rxa01436 1314 GBHTG3:AC010207 GBBA2:AF109682 GBHTG2:AC006759 GBHTG2:AC006759 GBBA1:MTY2OB11

GBBAI:XANXANAB

GBGSSIO:AQ194038 GBBA1:MTY2OB11 GBj3SS3:BI 0037 GBJ3SS3:B09549 GB-BAI:MTCY71 GBHTG5:AC007547 GB-HTG5:AC007547 GBBA2:AF072709 GB-BA1 :CGLYSEG GBPR4:AC005906 GB-BA1 :CGPTAACKA GBBA1:D90861 GB-PAT:E02087 unordered pieces.

990 AF109682 Aquaspirillum arcticum malate dehydrogenase (MDH) gene, complete cds. Aquaspirillumn arcticum 103725 AC006759 Caenoriiabdilis elegans clone Y40012, -SEQUENCING IN PROGRESS-, Caenorhabdltis elegans 8 unordered pieces.

103725 AC006759 Caenorhabditis elegans clone Y40G12, SEQUENCING IN PROGRESS-. Caenorhabditis elegans 8 unordered pieces.

36330 Z95121 Mycobacterium tuberculosis H37Rv complete genome: segment 139/162. Mycobacterium tuberculosis 3410 M8323 1 Xanthomon 'as campestris phosphoglucomutase and phosphomannomutase Xanthomonas campestris (xanA) and phosphomnannose isomerase and GDP-mannose pyrophosphorylase (xanB) genes, complete cds.

697 A01 94038 RPCI1 1 -47D24.TJ RPCI-1 1 Homo sapiens genomic clone RPCI-1 1-47D24, Homo sapiens genomic survey sequence.

36330 Z95121 Mycobaclerium tuberculosis H37Rv complete genome; segment 139/162. Mycobacterium tuberculosis 974 B10037 T27A19-T7 TAMU Arabidopsis thaliana genomlc clone T27A19, genomic Arabidopsis thaliana survey sequence.

1097 B09549 T21A19-T7.1 TAMU Arabidopsis Ihaliana genomlc clone T21A1 9, genomic Arabidopsis thaliana survey sequence.

42729 Z92771 Mycobacterium tuberculosis H-37Rv complete genome; segment 141/162. Mycobacterium tuberculosis 262181 AC007547 Homo sapiens clone RP1 1-252018, WORKING DRAFT SEQUENCE. 121 Homo sapiens unordered pieces.

262181 AC007547 Homo sapiens cdone RPi 1-252018, WORKING DRAIFT SEQUENCE, 121 Homo sapiens unordered pieces.

8366 AF072709 Streptomyces lividans amplifiable element AUD4: putative Streptomyces lividans transcriptional regulator. putative ferredoxin, putative cytochrome P450 oxidoreductase, and putative oxidoreductase genes, complete cds; and unknown genes.

2374 X96471 C.glutamicum lysE and lysG genes. Corynebacteriumn glutamicum 185952 AC005906 Homo sapiens l2pl 3.3 BAC RPCI1 1-429A20 (Roswell Park Cancer Homo sapiens Institute Human BAC Library) complete sequence.

3657 X89084 C.glutamicum pta gene and ackA gene. Corynebaclerlum glutamicumn 14839 090861 Ecoli genomic DNA, Kohara clone #405(52.0-52.3 min.). Escherichia coli 1200 E02087 DNA encoding acetate klnase protein form Escherlchia ccli. Escherichia coli 280 U60627 Helicobacter pylori feoB-like DNA sequence, genomic survey sequence. Helicobacter pylori 349 A170169i we8IcO4.xl SoaresNFLTGBC_51 Homo sapiens cDNA clone Homo sapiens IMAGE:2347494 3 similar to gb:L19686jrnal MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);, mRNA sequence.

207890 AC010207 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-92 25-F eb-99 17-Ju 1911 26-Apr-93 20-Apr-99 17-Jun-98 14-MAY-i 997 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 rxa01468 948 GB-GSS1:HPU60627 GBEST31 :A1701691 2007203041 29 Jun 2007 rxa01478 1959 rxa01482 1998 GB-EST15:AA480256 GB-BA1:SCI1 GB-BA1:SCE36 GB-BA1 :CGU43535 GBBA1:SC6G4 GBBAI:U00020 GBBA1:MTCY77 389 40745 12581 2531 41055 36947 22255 AL109848 AL049763 U43535 AL03 1317 U00020 Z95389 Table 4 (continu-ed) AA480256 nie3lfO4.sl NCICGAPCo3 Homo sapiens cIDNA clone IMAGE:898975 3' similar to gb:L19686 rnal MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);. mRNA sequence.

Streptomyces coelicolor cosmid 151.

Streptomyces coelicolor cosmId E36.

Corynebacterlum glutamicum multidrug resistance protein (cmr) gene, complete cds.

Streptomyces coelicolor cosmid 6G4.

Mycobacteriumn leprae cosmld 8229.

Mycobacterfum tuberculosis H37Rv complete genome; segment 146/1 62.

Homo sapiens Streptomyces coelicolor A3(2) Streptomyces coelicolor Corynebacterlum glutamicum Stheptomyces coelicolor Mycobacterlum leprac Mycobacterium tuberculosis 39,574 14-Aug-97 54.141 38.126 41,852 62,149 38.303 38,179 16-Aug.99 05-MAY-i 999 9-Apr-97 20-Aug-98 01-MAR-1994 18-Jun-98 rxa01 534 rxaOl535 1530 rxa01550 1635 GB-BAI :MLCB 1222 GBBA1:M1VO17 GB-BA1:PAU72494 GBBA1:D90907 GB_1N2:AF073177 GB_1N2:AF073179 34714 67200 4368 132419 9534 3159 AL049491 AL021897 U72494 D90907 AF073177 AF073179 Mycobaclerium leprae cosmid B1222. Mycobacterium leprae Mycobactedrlu tuberculosis H37Rv complete genome; segment 48/162. Mycobacteriurn tuberculosis Pseudomonas aeruginosa fumarase (tumO) and Mn superoxide dismulase Pseudomonas aeruginosa (sodA) genes, complete cds.

Synechocyslis sp. PCC6803 complete genome, 9/27, 1056467-1188885. Synechocystis sp.

Drosophila melanogaster glycogen phosphorylase (GlyP) gene, complete cds. Drosophila melanogaster Drosophila melanogaster glycogen phosphorylase (Gipi) mRNA, complete cds. Drosophila melanogaster 66,208 38,553 52,690 56,487 55,100 56,708 27-Aug-99 24-Jun-99 23-OCT-I1996 7-Feb-99 1-Jul-99 27-Apr-99 rxaQl 562 rxaOl 569 1482 GB BAI :078182 GBBA2:AF079139 GBBA2:AF087022 978 GB-BA1:MTCY63 GB-BA2:AF09751 9 7836 4342 1470 38900 4594 D78182 AF079 139 AF087022 Z96800 AF09751 9 Streptococcus mutans DNA for dIDP-rhamnose synthesis pathway, complete cds.

Streptomyces venezuelae pikCD operon, complete sequence.

Streptomyces venezuelae cytochrome P450 monooxygenase (pick) gene.

complete cds.

Mycobacterium tuberculosis H37Rv complete genome, segment 16/162.

Klebsiella pneumonlae dTDP-D-glucose 4.6 dehydratase (rmlB), glucose- Iphosphate thymidylyl transferase dTDP-4-keto-L-rhamnose reductase (rmlD). dTOP-4-keto-6-deoxy-D-glucose 3,5-epimerase (rmIC). and rhamnosyl transferase (wbbL) genes, complete cds.

Streptococcus mutans Streptomyces venezuelae Streptomyces venezuelae Mycobacterium tuberculosis Klebslella pneumoniae 44,050 38,587 38,621 59,035 59.714 5-Feb-99 28-OCT-I1998 15-OCT- 1998 17-Jun-98 4-Nov-98 2007203041 29 Jun 2007 Table 4 (continued) rxaOI571 723 GBBA1:AB01 1413 GBBAl:ABO1 1413 nxa01572 615 GB-BA1:A30 11413 GB_8Al:AB01 1413 8905 L091 89 Neisseria meningitidis dTDP-D-nlucose 4 .6-dehydratase (rfbB) olucose-I- Neisswrin meningilidis phosphate thymidyt transferase (rfbA) and rfbC genes, complete cds and UPDglucose-4-epimefase (galE) pseudogene.

12070 ABO11413 Streptomyces griseus genes for 0rf2. 00., 044, 045, AfsA, 04f8, partial and Streptomyces griseus complete cds.

12070 AB0l 1413 Streptomyces griseus genes for Orf2, 0r0, Orf4, 04f5, AfsA, 048B, partial and Streptomyces griseus complete cds.

12070 AB301 1413 Streptomyces griseus genes for 04f2, 04f3, 04f4, 04(5, AfsA, 04f8. partial and Streptomyces griseus complete cds.

12070 AB0l 1413 Streplomyces gdseus genes for 04f2. 043. 044. 04-5, AfsA, 048B. partial and Streptomyces griseus complete cds.

4783 U72240 Choristoneura tumniferana nuclear polyhedrosts virus ETM protein homolog, 79 Choristoneura fumiferana SA38 'I 0-Jnuhl-96 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 rxa01606 2799 GB_-Vl:CFU72240 GBGSS1O:AQ213248 GBGSS8:AQ070145 rxa01626 468 GBPR4:AF152510 GBPR4AF152323 GBPR4:AF1525J9 rxa01632 1 128 GB-HTG4:AC006590 GBHTG4:AC006590 kDa protein homolog. 15 kDa protein homolog and GTA protein homolog nucleopolyhedr genes, complete cds.

408 A0213248 HS_3249_BiAO2-MR CIT Approved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomic clone Plate=3249 Col=3 Row=B, genomic survey sequence.

285 A0070145 HS_3027_Bi_H02,MR CIT Approved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomlc clone Plate=3027 Coi=3 Row=P, genomic survey sequence.

2490 AF152510 Homo sapiens protocadherin gamma A3 short form protein (PCDH-gamma-A3) Homo sapiens variable region sequence, complete cds.

4605 AF152323 Homo sapiens protocadherin gamma A3 (PCDH-gamma-A3) mRNA, complete Homo sapiens cds.

2712 AF152509 Homo sapiens PCDH-gamma-A3 gene, aberrantly spliced, mRNA sequence. Homo sapiens 127171 AC006590 Drosophila melanogaster chromosome 2 clone BACR1 3NO2 (0543) RPCI-98 Drosophila mel 1 3.N.2 map 36E-36E strain y: cn bw sp, SEQUENCING IN PROGRESS-, 101 unordered pieces.

127171 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.

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

208780 Z99112 Bacillus subtilis complete genome (section 9 of 21): from 1598421 to 1807200. Bacillus subtilis 208780 Z99112 Bacillus subtilis complete genome (section 9 of 21): from 1598421 to 1807200. Bacillus sublilis 174368 AC006247 Drosophila melanogasler chromosome 2 clone BACR48110 (0505) RPCI-98 Drosophila mel 48.1.10 map 49E6-49F8 strain y; cn bw sp, SEQUENCING IN PROGRESS 17 unordered pieces.

ovirus 34,559 18-Sep-98 40,51 -Au-98 0 40,31 5Aug-8 anogaster 34,298 34,298 34,298 33.812 anogaster 33,812 14-Jul-99 22-Jul-99 14-Jul-99 19-OCT- 1999 19-OCT-1999 26-Jun-98 26-Nov-97 26-Nov-97 2-Aug-99 rxa01633 1206 GBGSS8:B99182 GBBAt:BSUBOO9 GBBAI:BSUBOOO9 GB-ITG2:AC006247 anogaster 36,111 36,591 34.941 37,037 2007203041 29 Jun 2007 Table 4 (Continued) Corynebacterium glutamicum DNA for L-Malate:qulnone oxidoreductase.

rxa01695 1623 GBBA1:CGA224946 GBBA1:MTCY24A1 GBIN1:DMU15974 rxaOl17O2 1155 GBBA1:CGFDA GBBA1:MTYI3E1O GBBA1:MLCB4 rxa01743 901 GB_1N2:CELC27H5 GBEST24:A1167112 GBGSS9:A0102635 rxa01744 1662 GBBA1:MTCYO1B2 GBGSS1I:AF009226 GB-BA1 :SCD78 rxa01745 836 GBBAI:MTCYI9O GBBAI:MLCB22 GB 8A2:AE0OO175 rxa01758 1140 GBPR3:HS57G9 GB-PL2:YSCH9666 GBPL2:YSCH9986 rxa01814 1785 GBBAI:ABCCELB GBBA1:MTCY22D7 GBBA1:MTCY22D7 rxaOlB51 1809 GB-GSS9:AQ142579 GB_1N2:AC005889 GBGSSI:AGOOS8I4 2408 AJ224946 Corynebacterium 100,000 11 1-Aug-98 20270 2994 3371 35019 36310 35840 579 347 35938 665 36224 34150 40281 15067 113872 39057 41664 2058 31859 31859 529 108924 637 Z95207 U 15974 X17313 Z95324 AL02 35 14 U 14635 At 167 112 AQ102635 Z95554 AF009226 AL034355 Z70283 Z98741 AE000 175 Z95116 U 10397 U00027 L24077 Z83866 283866 AQ 142579 AC005889 AGO0881 4 glutamicum Mycobacterium tuberculosis H37Rv complete genome; segment 1241162. Mycobacterium tuberculosis Drosophia melanogaster kinesin-like protein (ktp6Bd) mRNA, complete cds. Drosophila metanogaster Corynebacterlum glutamicum fda gene for fructose-bisphosphate aldolase (EC Corynebacterium 4.1.2.13). glutamlcum' Mycobacterlum tuberculosis H37Rv complete genome; segment 18/162. Mycobacterium tuberculosis Mycobacterium leprae cosmid B4. Mycobacterlum leprae Caenorhabditls elegans cosmid C27H5. Caenorhabditis elegans xylem.esl.878 Poplar xylem Lambda ZAPII library Poputus balsamifera subsp. Poputus balsamifera trichocarpa 6DNA mRNA sequence. subsp. trlchocarpa HS 73O48-B1FO8_MF CIT Approved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomic clone Plate=3048 001=1 5 Row=L, genomic survey sequence.

Mycobaclerlum tuberculosis H37Rv complete genome; segment 72/162. Mycobacterium tuberculosis Mycobacterium tuberculosis cytochrome D oxldase subunit I (appO) gene, Mycobacterium partial sequence, genomlc survey sequence. tuberculosis Streptomyces coelicotor cosmid D78. Streptomyces coelicolor Mycobacterium tuberculosis H37Rv complete genome; segment 98/162. Mycobacterium tuberculosis Mycobacterium leprae cosmid B22. Mycobacterium lepre Escherlchla coll K-12 MG1655 section 65 of 400 of the complete genome. Escherlchia col Human DNA sequence from BAG 57G9 on chromosome 22q 12.1 Contains Homo sapiens ESTs, CA repeat, GSS.

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 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 cerevislae chromosome ViII cosmid 9666. Saccharomyces cerevislae 40,021 Saccharomyces cerevislae chromosome ViII cosmid 9986. Saccharomyces cerevislae 34,375 Acetobacter xylinum phosphoglucomutase (ceiB) gene, complete cds. Acetobacter xylinus 62,173 Mycobacterium tuberculosis H37Rv complete genome: segment 133/162. Mycobacterium 39,749 tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 133/162. Mycobacterium 40,034 tuberculosis HS_72222_Bi_H03_MR CIT Approved Human Genomic Sperm Library D Homo Homo sapiens 38,068 sapiens genomic clone Plate=2222 CoI=5 Row=P. genomic survey sequence.

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

Homo sapiens genomic DNA, 21q region, clone: D137B7BB68. genomic survey Homo sapiens 35,316 sequence.

2007203041 29 Jun 2007 rxa01859 1050 GBBA2:AF183408 GB-HTG5:AC008031 GB_2A2:AF183408 rxa01865 438 GB-BA1:SERFDXA GB-BA11:MTVOO5 GBBA1:MSGY348 rxa01882 1113 GBPR1:-UMADRA2C GB-PR4:HSU72648 GB-GSS3:B42200 63626 AF183408 156889 AC008031 63626 AF183,408 rxa01884 1913 GBBA1:MTCY48 GBBA1:SC0001206 GBBA1:D90908 rxa01886 897 GBGSS9:AQI 16291 GB-BA2:AE001721 GBEST16:MA567090 rxa018Bl 1134 GB-HTG6:AC008147 GBHTG6:AC008147 GB-BA2:ALW24343 1 3869 37840 40056 1491 4850 387 35377 9184 122349 572 17632 596 303147 303147 26953 M61 119 AL0 10186 AD000020 J03853 U72648 B42200 Z74020 AJO01 206 090908 AQ 1162 91 AE001 721 AA567090 AC008147 AC008 147 AJ243431 Table 4 (continued) Microcystis aeruginosa DNA polymerase III beta subunit (dnaN) gene, partial cds; microcystin synthetase gene cluster, comptete sequence; Umal (umal1).

Uma2 (uma2), Uma3 (uma3), Uma4 (uma4), and Uma5 (uma5) genes, comptete cds; and Uma6 (uma6) gene, partial cds.

Trypanosoma brucei chromosome 11 ctone RPCI93-25N14, SEQUENCING IN PROGRESS 2 unordered pieces.

Microcystis aeruginosa DNA potymerase III beta subunit (dnaN) gene, partial cds; mlcrocystln synthelase gene cluster, complete sequence; Umal (umal), Uma2 (uma2), Uma3 (uma3), Uma4 (uma4), and Uma5 (uma5) genes, complete cds; and Uma6 (uma6) gene, partial cds.

Saccharopotyspora erythraea ferredoxin (tdxA) gene, complete cds.

Mycobacter tuberculosis H37Rv complete genome: segment 51/162.

Mycobacterium tuberculosis sequence from clone y348.

Human kidney atpha-2-adrenergic receptor mRNA, complete cds.

Homo sapiens alpha2-C4-adrenerglc receptor gene, complete cds.

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

Mycobactedrn tuberculosis H37Rv complete genome: segment 69/162.

Streptomyces coeticolor A3(2), glycogen metabolism cluster 11.

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

RPCII1 1-49P6.TK.1 RPCI-1 1 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 metanogaster ovary BlueScript Drosophila melanogaster cDNA clone GM01044 5prime, mRNA sequence.

Homo sapiens clone RP3AOSJ1O, SEQUENCING IN PROGRESS ,102 unordered pieces.

Homo sapiens clone RP3-405J1 10, SEQUENCING IN PROGRESS ',102 unordered pleces.

Aclnetobacter Iwofii wzc, wzb, wza, weeA, weeB, wceC, wzx, wzy. weeD, weeE, weeF, weeG, weeH, weel, weeJ, weeK, galU, ugd. pgi, galE, pgm (partial) and mlp (partial) genes (emulsan blosynthetic gene cluster), strain RAG-i1.

Microcystis aeruginosa 36,364 Trypanosoma brucei 35.334 Microcyslis aeruginosa 36,529 Saccharopolyspora 59,862 erythraea Mycobacteriumn 61 .949 tuberculosis Mycobacterium 59,908 tuberculosis Homo sapiens 36,899 Homo sapiens 36,899 Homo sapiens 34.805 Mycobacterium 37,892 tuberculosis Streptomyces coelicolor 40,413 Synechocystis sp. 47,792 Homo sapiens 43,231 Thermotoga maritima 39,306 Drosophila melanogaster 42,807 Homo sapiens 36,417 Homo sapiens 37,667 Acinelobacter Iwoffli 39,640 Drosophila melanogaster 32,969 03-OCT.1999 15-Nov-99 03-OCT-i1999 1 3-MAR-1996 17-Jun-98 10-DEC-1996 27-Apr-93 23-Nov-98 18-OCT-i1997 1 7-Jun-98 29-MAR-i 999 7-Feb-99 20-Apr-99 2-Jun-99 28-Nov-98 03-DEC-i 999' 03-DEC-i1999 01 -OCT-1999 2-Aug-99 rxa01888 658 GB-HTG2:AC008197 125235 AC008197 Drosophila melanogaster chromosome 3 clone BACRO2L-12 (D753) RPCI-98 021.12 map 94B-94C strain y; cn bw sp, -SEQUENCING IN PROGRESS-, 113 unordered pieces.

2007203041 29 Jun 2007 GBHTG2:AC008197 GBEST36:A1881527 rxaOl891 887 GB-VI:H1V232971 GBPL1:AFCHSE GBPR3:AFO64858 nM01895 1051 GBBA1:CGL238250 GBBA2:AF038423 GB_8A1:MTCY359 rxa0l90I 1383 GBBA1:MSGB3BCOS GBBAI:SCE63 GBPR3:AF093117 ra01927 1503 GBBA1:CGPAN GBBA1:ASXYLA GBHTG3:AC009500 rxa01952 1836 GBBA2:AE000739 GBEST28:AI519629 GBEST2I:AA949396 rxa01989 630 GBBA1:BSPGIA GB-BA1 :BSUBOO1 7 GBBA2:AF132127 rxa02O26 720 GBBA1:SXSCRBA GSB Al :BSUB0O2O GB-BA1 :BSGENR rxa02028 526 GBBA1:MTC1237 125235 AC008197 Table 4 (continued) Drosophila melanogaster chromosome 3 clone BACRO2LI12 (0753) RPCI-98 Drosophila melanogaster 32.969 021L.12 map 94B3-94C strain y; cn bw sp, SEQUENCING IN PROGRESS -,113 unordered pieces.

606070C09.y 1 606 Ear tissue cDNA library from Schmidt lab Zea mays cDNA, Zea mays 43,617 2-Aug-99 598 621 61I58 193387 1593 1376 36021 37114 37200 147216 2164 1905 176060 13335 612 767 1822 217420 8452 3161 212150 97015 27030 A1881 527 AJ232971 Y09542 AF064858 AJ238250 AF038423 Z83859 L01095 AL035640 AF0931 17 X96580 X59466 AC009500 AE000739 A151 9629 AA94 9396 X16639 Z99120 AF132127 X67744 Z991 23 X73124 Z94752 mRNA sequence.

Human immunodeficiency virus type 1 subtype C net gene, patient MP83. Human immunodeficiency virus type I Afumigatus chsE gene. Aspergiltus fumigatus Homo sapiens chromosome 21q22.3 BAC 28F9, complete sequence. Homo sapiens Corynebaclerium glutamlcum ndh gene. Corynebacterlum glutamicum Mycobacteriunm smegmatis NADH dehydrogenase (ndh) gene, complete cdls. Mycobacterlum smegmatis Mycobacterium tuberculosis H37Rv complete genome; segment 84/162. Mycobacterum tuberculosis M. teprae genomic DNA sequence. cosmid B38 btr gene, complete cdls. Mycobacterium leprae Streptomyces coelicolor cosmid E63. Streptomyces coelicolor Homo sapiens chromosome 7qtelo BAG E3, complete sequence. Homo sapiens C.glutamicum panB, panG xylB genes. Corynebacterium glutamlcum Arthrobacter Sp. N.R.R.L. 63728 xytA gene for D-xylose(D-glucose) isomerase. Arthrobacter sp.

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

Aquifex aeollcus section 71 of 109 of the complete genome. Aquifex aeolicus LD39282.5prlie LD Drosophila melanogaster embryo pOT2 Drosophila Drosophila melanogaster melanogaster cONA clone LD39282 5prime, mnRNA sequence.

LD28277.Sprime LD Drosophila melanogaster embryo pOT2 Drosophila Drosophila melanogaster melanogaster cDNA clone LD28277 5prlme, mnRNA sequence.

Bacillus stearothermophilus pgiA gene for phosphoglucolsomerase isoenzyme Bacillus A (EC stearothermophilus Bacillus subtilis complete genome (section 17 of 21): from 3197001 to Bacillus subtilis 3.414420.

Streptococcus mutans sorbilol phosphoenotpyruvate:sugar phosp hotransf(erase Streptococcus mutans operon. complete sequence and unknown gene.

S.xytosus scrB and scrR genes. Staphylococcus xylosus Bacillus subtilis complete genome (section 20 of 21): from 3798401 to Bacillus sublilis 4010550.

8.subilis genomic region (325 to 333). Bacillus subtills Mycobacterlum tuberculosis H37Rv complete genome; segment 46/162. Mycobacterium tuberculosis 40,040 37,844 37,136 100,000 65,254 40,058 59,551 39,468 39,291 38,384 56,283 37,593 36,309 41,941 39,855 66,292 37,255 63.607 67,778 35,574 51,826 54,476 21-Jul-99 05-MAR-1999 1 -Apr-97 2-Jun-98 24-Apr-99 05-MAY-i 998 17-Jun-98 6-Sep-94 117-MAR-I 999 02-OCT-i1998 11 -MAY- 1999 04-MAY- 1992 24-Aug-99 25-MAR-i1998 116-MAR-i 999 25-Nov-98 20-Apr-95 26-Nov-97 28-Sep-99 28-Nov-96 26-Nov-97 2-Nov-93 17-Jun-98 2007203041 29 Jun 2007 rxa02054 1140 rxa02O56 2891 r=02061 1617 rxa02O63 1350 rxa02100 2348 rxaO2122 822 rxaO2l4O 1200 rxa02l42 774 GBPL2:SCE9537 GBGSS13:AQ501177 GBBAI:MLCB1222 GBBA1:MTY13E12 GB-BA1 :MTU43540 GBPAT:E14601 GBBA1:D8,4102 GB-BA1:MTVOO6 GBHTG7:AC005883 GBPL2:ATAC003033 GBPL2:ATAC002334

GBBAI:SCGLGC

GB-GSS4:AQ687350 GBEST38:AW028530 GB-BAI:MSGY1 51 GB-BA1 :MTCY1 30 GB-BA1 :SC0001205 GBBA1:D90858 GBEST37:A1948595 GB-HTG3: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 E14 601 084102 ALO21 006 ACOD5883 AC003033 AC002334 X89733 AQ687350 AW02 8530 AD00001 8 Z73902 AJO01 205 090858 A1948595 AC010387 L78813 L7881 4 AF093099 Z70283 Table 4 (continued) Saccharomyces cerevislae chromosome V cosmids 9537, 9551. 9495, 9867, Saccharomyces cerevisiae 36,100 and lambda clone 5898.

V26G9 mTn-3xHAacZ Insertion Library Saccharomyces cerevisiae genomic Saccharomyces cerevisiae 32,039 genomic survey sequence.

Mycobacterium Ieprae cosmid B 1222. Mycobacterium Ieprae 61,896 27-Aug-99 1 -Aug-97 29-Apr-99 Mycobaclerium tuberculosis I-37Rv complete genome; segment 1471162. Mycobacterium tuberculosis Mycobacterium tuberculosis rfbA, rhamnose biosynthesis protein (nfbA), and Mycobacterium YmlC genes, complete cds. tuberculosis Brevibacterium laclotermentum gene for alpha-ketoglutadc acid Corynebacterium dehydrogenase. glulamicum Corynebacterlum glutamicum DNA for 2-oxoglutarate dehydrogenase. complete Corynebacterium cds. glutamicum Mycobacterium tuberculosis H37Rv complete genome; segment 54/162. Mycobacterium tuberculosis Homo sapiens chromosome 17 clone RP1 1-958El11 map 17. Homo sapiens SEQUENCING IN PROGRESS 2 ordered pieces.

Arabidopsis thaliana chromosome 11 BAC T211-14 genomic sequence, complete Arabidopsis thaliana sequence.

Arabidopsis thaliana chromosome 11 BAC F25118 genomic sequence, complete Arabidopsis thaliana sequence.

S.coelicolor DNA for glgC gene. Streptomyces coelicolor nbxbOO74H-1 1ir CUGI Rice BAC Library Oryza sativa genomic clone Oryza sativa nbxbOO74Hi 1 r. genomic survey sequence.

wv27flO.xl NCI CGAP Kidi 1 Homo sapiens cDNA clone IMAGE:2530795 3' Homo sapiens similar to WP:TO3G1 1.6 CE04874 mRNA sequence.

Mycobacterium tuberculosis sequence from clone y15l. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genomne; segment 59/162. Mycobacterium tuberculosis Streptomyces coelicolor A3(2) glycogen metabolism cdusterd. Streptomyces coelicolor Ecoli genomic. DNA. Kohara clone #401(51.3-51.6 min.). Escherichla coli wqO7dl2.xl NOICGAPKid12 Homo sapiens cDNA clone IMAGE:2470583 Homo sapiens mRNA sequence.

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

Mycobacterium leprae cosmid B 1551 DNA sequence. Mycolbacterium leprae Mycobacterium Ieprae cosmid 81554 DNA sequence. Mycobacterium Ieprae Mus musculus transcription factor TBLYM (Tblym) mRNA, complete cds. Mus muscutus Mycobacterium tuberculosis 1437Rv complete genome; segment 98/1 62. Mycobacterium tuberculosis 59,964 59,659 98.928 98,928 39,265 37,453 37,7 11 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 57,292 117-Jun-98 14-Aug-97 28-Jul-99 6-Feb-99 18-Jun-98 08-DEC-1999 19-DEC-1997 04-MAR-1998 c 12-Jul-9 1-Jul-99 27-OCT-I1999 10-DEC-1996 17-Jun-98 29-MAR-i 999 29-MAY-1 997 6-Sep-99 15-Sep-99 15-Jun-96 15-Jun-96 01-OCT-i1999 17-Jun-98 GBBA1:MSGB1S51CS 36548 GBBAI:MSGB1554CS 36548 GBRO:AF093099 2482 GB-BA1:MTCY190 34150 2007203041 29 Jun 2007 Table 4 (continued) 36734 AL049497 Streptomyces coelicolor cosmid 6Glb. GBBA1:SC6G1O 68_BA1:AB016787 5550 nxa02143 1011 GBBA1:MTCY19O 34150 GBBA1:MSGB15S1CS 36548 GB-BA1:MSGB1554CS 36548 nxa02l44 1347 GBBA1:MTCYI9O 34150 GBHTG3:ACO1I500_0300851 GB-HTG3:AC01 15000 300851 rxa02147 1140 GBEST28:A1492095 GBEST1O:MA157467 GBEST1O:AA157467 ra02149 1092 GBPR3:HSBK277P6 GBBA2:EM8065R075 GBEST34:Al789323 rxaO2l75 1416 GBBA1:CGGLTG GBBAI:MTCY31 GBBA1:MLCB57 rxa02196 816 GBRO:RATDAPRP GBGSS8:AQ012162

GB-RO:RATDAPRP

rxa02209 1694 GBBAI:AF3025424 G(3_BA2:AF002133 485 376 376 61698 360 574 3013 37630 38029 2819 763 2819 2995 15437 ABO 16787 Z70283 L78813 L78814 Z70283 AC0I 1500 AC0 11500 A1492095 AA1 57467 AM1 57467 AL1 17347 AF 116423 A1789323 X66 112 Z731 01 Z99494 M76426 AQ012 162 M76426 AB025424 AFOO2I 33 Pseudornonas putida genes for cytochrome o ubiqulnol oxidase A-E and 2 ORFs, complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 98/162.

Mycobacterium Ieprae cosmid B1551 DNA sequence.

Mycobacterium leprae cosmid B1554 DNA sequence.

Mycobacterium tuberculosis H37Rv ,complete gerlome; segment 98/1 62.

Homo sapiens chromosome 19 clone CIT978SKB_60E1 I i, SEQUENCING IN PROGRESS 246 unordered pieces.

Homo sapiens chromosome 19 clone C1T978SKB-60E1 1. SEQUENCING IN PROGRESS 246 unordered pieces.

tgO7a0l.xl NCl-CGAP.CLL1 Homo sapiens cONA clone IMAGE:2108040 3, mRNA sequence.

zo50e0l.rl Stralagene endothelial cell 937223 Homo sapiens cDNA clone IMAGE:590328 5% mRNA sequence.

zo50eOI.rl Stratagene endothelial cell 937223 H-omo sapiens cONA clone IMAGE:590328 mRNA sequence.

Human DNA sequence from clone 277P6 on chromosome 1 q25.3-31.2.

complete sequence.

Rhizobium etli mutant MB045 RcsR-transcriptionally regulated sequence.

uk53g05.yl Sugano mouse kidney mkia Mus musculus cONA clone IMAGE:1972760 5 similar to WP:K1 11-112.8 CE12160 mRNA sequence.

C.glutomicum glt gene for citrate synthase and ORF.

Mycobacterium tuberculosis H37Rv complete genome: segment 41/162.

Mycobacterium leprae cosmid B57.

Rattus norvegicus dipeptidyl amino peptidase- related protein (dpp6) mRNA, complete cds- 127P8037070197 Cosmid library of chromosome 11 Rhodobacter sphaeroides genomic clone 127PB037070197, genomlc survey sequence.

Rattus norvegicus dipeplldyl aminopeplldase-related protein (dpp6) mRNA.

complete cds.

Corynebacterium glutamicum gene for aconilase. partial cds.

Mycobacterium avium strain GIRIO transcriptional regulator (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.

Streptomyces coelicolor Pseudomonas pulida Mycobacterium tuberculosis Mycobacterium leprae Mycobacterium Ieprae Mycobacterium tuberculosis Homo sapiens Homo sapiens Home sapiens Homo sapiens Home sapiens Home sapiens Rhizobium etli Mus musculus Corynebacterlum glutamicum Mycobacterium tuberculosis Mycobacterium Ieprae Rattus norvegicus Rhodobacter sphaeroides Rattus norvegicus Corynebacterlum glutamicum 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-i1999 5-Aug-99 17-Jun-98 15-Jun-96 17-Jun-98 18-Feb-00 1 8-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 2007203041 29 Jun 2007 GB.BA1:MTV007 rxa022i3 874 GBBA1:AB025424 GBBA1:MWV007 GBBA2:AF002133 rxa02245 780 GB-BA2:RCU23145 32806 2995 32806 15437 AL021 1114 A8025424 AL02 1184 AF0021 33 Table 4 (continued) Mycobacterium tuberculosis H37Rv complete genome; segment 64/162. Mycobacterium tuberculosis Corynebacterium glutamicum gene for aconitase. partial cds. Corynebacterium glutamicum Mycobacterium tuberculosis H37Rv complete genome; segment 641162. Mycobacteriurn tuberculosis Mycobacterium avlumn strain GIRIO transcriptional regulator (may81) gene. Mycobacterium avium, partial cds, aconitase (acn), invasin i (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 capsulatus 1,6-/sedohe ptulos e- 1.7-bisph osph ate aldolase (cbbA) gene, partial cds. Form 11 ribulose-1 ,5-bisphosphate carboxylase/oxygenase (cbbM) gene, complete cds, and Calvin cycle operon: pentose-5-phosphate-3-epimerase (cbbE), phosphoglycolate phosphatase (cbbZ), and cbbY genes, complete cds.

38.253 99,096 34.937 36,885 48,701 17-Jun-98 3-Apr-99 17-Jun-98 26-MAR-1 998 28-OCT-i1997 5960 U23145 GB BA 1:ECU82664 GBHTG2:AC007922 rxa02256 1125 GB-BA1:CGGAPPGK GB-BA1 :SCC54 GBBA1:MTCY493 rxa02257 1338 GBBAI:CGGAPPGK GB BAt :MTCY493 GBBA2:MAU82749 rxa02258 900 GBBAI:CGGAPPGK GD-BA1 :CORPEPC GByPAT:A09073 rxa02259 2895 GBBAl:CORPEPC GBPAT:A09073 GB-BA1:CGPPC 139818 158858 3804 30753 40790 3804 40790 2530 3804 4885 4885 4885 4885 3292 U82664 AC007922 X59403 AL035591 Z95844 X59403 Z95844 U82749 ?C59403 M25819 A09073 M25819 A09073 X14234 Escherichia coli minutes 9 to 11 genomic sequence.

Homo sapiens chromosome 18 clone hRPK178 F 0 map 18, SEQUENCING IN PROGRESS 11 unordered pieces.

C.glutamicum gap, pgk and tpi genes for glyce ralIdehyde-3-phosph ate, ph osphoglyce rate kinase and triosephosphate Isomerase.

Streptomyces coelicolor cosmid C54.

Mycobacterium tuberculosis H37Rv complete genome; segment 63/162.

Cglutamlcumn gap, pgk and tpl genes for glyceraldehyde-3-phosphate.

phosph oglyce rate kinase and triosephosphate Isomerase.

Mycobacterium tuberculosis H37Rv complete genome; segment 63/1 62.

Mycobacterium avium glyceraldehyde-3-phosphate dehydrogenase homolog (gapdh) gene, complete cds; and phosphoglycerate kinase gene, partial cds.

C.glutamicum gap. pgk and tpi genes for glyceralde hyde-3- phosph ate, phosphog lyce rate kinase and triosephosphate isomerase.

Cglutamicum phosphoenolpyruva te carboxylase gene, complete cds.

C.glutamicum ppg gene for phosphoenol pyruvale carboxylase.

C.glutamicum phosphoenolpyruvate carboxylase gene, complete cds.

C.glutamicum ppg gene for phosphoenol pyruvate carboxylase.

Corynebaclerium glutamicum phosphoenolpyruvate carboxylase gene (EC 4.1.1.31).

Escherichia coli Homo sapiens Corynebacerium glutamlicurn Streptomyces coelicolor Mycobacterium tuberculosis Corynebacterium glutamicum Mycobacterium tuberculosis Mycobacterium avium Corynebacterlurn glutamicum Corynebacterlum glutamicum Corynebacterlurn glutamicum Corynebacterium glutamicum Corynebacterium glutamicum Corynebacterium glutamnicumn 39,119 11-Jan-97 99,289 36,951 64.196 98,873 61,273 61,772 99,667 100,000 10D0,000 100,000 100,000 05-OCT-I1992 11-Jun-99 19-Jun-98 05-OCT-i1992 19-Jun-98 6-Jan-98 05-OCT-1992 15-DEC-1995 25-Aug-93 15-DEC-1995 25-Aug-93 99,827 12-Sep-93 2007203041 29 Jun 2007 rxa02288 969 GBPR3:HSDJ94E24 GBHTG3:ACO100g1 GBHTG3:ACO10091 rxa02292 798 GBBA2:AF125164 GBGSS5:AQ744695 GB-EST14:AA381925 rxa02322 511 GB-BA1:MTCY22GB GBBAI:MTCY22G8 rxa02326 939 rxa02327 1083 rxa02328 1719 rxa02332 1266 fxa02333 1038

GBBAI:CGPYC

GBBA2:AF038548 GBBA1:MTCY349

GB-BAI:CGPYC

GBBA2:AF038548 GBBA1:MTCY349 GBBA1:CGPYC GBBA2:AF038548 GB PL2:AF097728 GBBA1:MSGLTA GB-BA2ABU85944 GB BA2:AE0001 75 GBBA1;MSGLTA 243145 159526 159526 26443 827 309 22550 22550 3728 3637 43523 3728 3637 43523 3728 3637 3916 1776 1334 15067 1776 AL05031 7 AC01O091 AC0 10091 AF125164 A0744695 AA381 925 Z95585 Z95585 Y09548 AF038548 Z83018 Y09548 AF038548 Z83018 Y09548 AF038543 AF097723 X60513 U85944 AE000175 X60513 Table 4 (continued) Human DNA sequence from dlone RPI-94E24 on chromosome 20ql2.

complete sequence.

Homo sapiens clone NI-10295A01, SEQUENCING IN PROGRESS 4 unordered pieces.

Homo sapiens clone NHO1295AOI, SEQUENCING IN PROGRESS 4 unordered pieces.

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

HS_-5505_-A2_-C06_SP6-RPCI-l1 Human Male BAC Library Homo sapiens genomloc clone Plate='1081 Col=12 Row--E. genomic survey sequence.

EST95058 Activated T-ceils I Homo sapiens cIDNA 5Tend, mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.

Corynebacterium glutamicum pyc gene.

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

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

Corynebacterlumn glulamicum pyc gene.

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

Mycobacterlumn tubercuiosis H37Rv complete genome: segment 131/162.

Corynebacterium glulamicum pyc gene.

Corynebacteriuim glulamicum pyruvate carboxylase (pyc) gene. compiete cds.

Aspergillus terreus pyruvate carboxylase (Pyc) mRNA. complete cds.

M.smegmatls gItA gene for citrate synthase.

Antarctic bacterium DS2-3R citrate synthese (cisy) gene, complete cds.

Escherlchia coli K-12 MG 1655 section 6501f400 of the comple genome.

M.smegmalls gltA gene for citrate synihase.

Homo sapiens Chromosome 16 BAC clone CIT987-SKA-1 13A6 -complete genomic sequence, complete sequence.

Homo sapiens Homo sapiens Homo sapiens Bacteroides fragilis Homo sapiens Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis Corynebacterium glutamicumn Corynebacterium glutarmlcum Mycobacterium tuberculosis Corynebacterium glutamicum Corynebacterlumn glutamicum Mycobaclerium tuberculosis Corynebacterium glutarnicum Corynebacterium glutamicum Aspergillus terreus 36.039 35, 33 1 35,331 39,747 39,185 35,922 57,677 37,143 100,000 100,000 37,363 99,259 99,259 4 1.317 100,000 100,000 03-DEC.1999 11I-Sep-99 11 -Sep-99 01-DEC-1999 16-Jul-99 21 -Apr.97 17-Jun-98 117-Jun-98 08-MAY-I1998 24-DEC-1997 17-Jun-98 08-MAY-1 998 24-DEC-I1997 17-Jun-98 08-MAY-I1998 24-DEC- 1997 52,248 29-OCT-1998 Mycobacterium smegmatis 58,460 Antarctic bacterium DS2- 57,154 3R Escherichla coi 38,164 Mycobacterium smegmatis 58,929 Homo sapiens 33,070 20-Se p- 9 1 23-Sep-97 12-Nov-98 20-Sep-91 23-Nov-99 GBPR4:HUAC002299 171681 AC002299 2007203041 29 Jun 2007 Table 4 (continued) Drosophila melanegaster chromosome 3 clone BACR48EI 2 (13695) RPCI-98 Drosophila melanogaster GBHTG2:AC007889 127840 AC007889 34,897 rxa02399 1467 GBBA1:CGACEA GB-BA1 :CORACEA GBPAT:113693 rxa02404 2340 GBBAI:CGACEB GBBA1:CORACEB GB-BAI:PFFC2 rxa02414 870 GBPR4:AC007102 GBMTG3:AC01 1214 GBHTG3;ACO I1214 rxa02435 681 GBBA2:AF101055

GBOM:RABPKA

GBOM:RABPLASISM

rxa02440 963 GBEST14:AA417723 GBESTli:AA215428 GBBAI:MTCY77 rxa02453 876 GBEST14:AA426336 GBBA1:STMAACC8 GBPR3:AC004500 rxa02474 897 GBBA1:AB009078 GB-OM:BTU71200 GB-EST2:F1 2685 rxa02480 1779 GB_BA1:MTVO12 2427 1905 2135 3024 2725 5588 176258 1834 14 183414 7457 4441 4458 374 303 22255 375 1353 77538 2686 877 287 70287 X75504 L28760 113693 X78491 L27123 Y11998 AC007102 AC01 1214 AC01 1214 AF101 055 J03247 M64656 AA417723 AA21 5428 Z95389 AA4 26336 M55426 AC004500 ABOO9078 U71200 F 12685 AL02 1287 48.E.12 map 87A-87B strain y; cn bw sp. -SEQUENCING IN PROGRESS-, 86 unordered pieces.

C.glutamicum aceA gene and thiX genes (partial).

Corynebacterium glutamicum isocitrate lyase (aceA) gene.

Sequence 3 from patent US 5439822.

C.glutamicum (ATCC 13032) aceB gene.

Corynebacterlum glutamicum malate synthase (aceB) gene, complete cds.

P.fluorescens FC2.1, FC2.2, FC2.3c, F02.4 and FC2.5c open reading frames.

Homo sapiens chromosome 4 clone C0162P16 map 4p16, complete sequence.

Home sapiens clone LOW-PASS SEQUENCE SAMPLING.

Memo sapiens clone 5_C_3, LOW-PASS SEQUENCE SAMPLING.

Clostridium acetobutylicum alp operon, complete sequence.

Rabbit phosphorylase kinase (alpha subunit) mRNA, complete cds.

Oryctolagus cuniculus phosphorylase kinase alpha subunit mRNA, complete cds.

zvO1Il2.sl NCICGAPGOBi Homo sapiens cDNA clone IMAGE:746207 3' similar to contains Alu repetitive element;contains element Li repetitive element:, mRNA sequence.

zr95a07.si NCI CGAP GOB1 Homo saplens cDNA clone IMAGE:683412 3' similar to contains Alu repetitive element:, mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genomo; segment 146/1 62.

zv53g02.sI Soares-testisNHT Homo sapiens cONA clone IMAGE:757394 3T, mRNA sequence.

S.fradlae aminoglycoside acetyltransferase (aacC8) gene, complete cds.

Homa sapiens chromosome 5. P1 clone 1076139 (LBNL H14). complete sequence.

Brevibacterium saccharolyticum gene for L-2.3-butanediol dehydrogenase, complete cds.

Bos taurus acetoln reductase mRNA, complete cds.

HSC3DAO31 normalized infant brain cDNA Homo sapiens cODNA clone c- 3da03, mRNA sequence Mycobacterium tuberculosis M37Rv complete genome; segment 132/1 62.

Cerynebacterlum 100,000 glutamicum Corynebacterium10,0 glutamicum Unknown. 99,795 Corynebacterlum 99,914 glutamicum Cerynebacterium 99,786 glutamicumn Pseudemonas fluorescens 63,539 Memo sapiens 35,069 Homo sapiens 36,885 Home sapiens 36,885 Clostridium acetobutylicumn 39,605 2-Aug-99 9-Sep-94 10-Feb-95 26-Sep-95 13-Jan-95 8-Jun-95 11I-J ul-97 2-Jun-99 03-OCT-i1999 03-OCT-i1999 03-MAR-i1999 Oryctelagus cuniculus Oryctolagus cuniculus Memo sapiens Home sapiens Mycobacterium tuberculosis Memo sapiens Streptomyces fradiae Memo sapiens Brevibacterlum sacoharelyticumn Bes taurus Home sapiens Mycobacterium tuberculosis 36,061 27-Apr-93 36,000 38,770 39,934 38,889 38,043 37,097 33,256 96,990 51,659 41,509 36.737 22-Jun-98 16-OCT-1 997 13-Aug-97 18-Jun-98 1 6-OCT-i 997 05-MAY-i1993 30-MAR-i1998 13-Feb-99 8-Oct-97 14-Mar-95 23-Jun-99 2007203041 29 Jun 2007 GBBA1:AP000060 nxa02485 rxa02492 840 GB_BAI:STMPGM GBBA1:MTCV2OG9 GB_BA1:U00018 rxa02528 1098 GBPR2:HS161NIa GB-HTG2:AC008235 36734 347800 921 37218 42991 56075 136017 AL049497 AP000060 M83661 Z77 162 UO0018 AL008707 AC008235 GBHTG2:AC008235 136017 AC008235 rxa02539 1641 GBBA2:RSU17129 GBBA1:MTVO38 GBBA2:AF068264 17425 16094 3152 rxaO2551 483 GBBAi:BACHYPTP 17057 GBBAl :BACHUThVAPQ8954 GB_BA1:BSGBGLUC 4290 rxa02556 1281 GBHTG3:AC008128 335761 GB-HTG3:AC008128 335761 GBPL2:AC005292 99053 rxaO2560 990 GBIN1:CEFO7A1 1 35692 GBE5T32:Al73i605 566 U 17129 AL021 933 AF068264 029985 031856 Z34526 AC008 128 AC008128 AC005292 Z6651 1 A1731605 Table 4 (continued) Streptomyces coelicolor cosmid 6G10.

Aeropyrum pernix genomic DNA, section 317.

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

Mycobacterium tuberculosis H37Rv complete genome; segment 25/1 62.

Mycobacterium Ieprae cosmid B2168.

Human DNA sequence from PAC 161N10 on chromosome Xq25. Contains

EST.

Drosophila melanogaster chromosome 3 clone BACR15B19 (0995) RPCI-98 15.13.19 map 94F-95A strain y; cn bw sp. -SEQUENCING IN PROGRESS -,125 unordered pieces.

Drosophila melanogaster chromosome 3 clone BACR15B319 (0995) RPCI-98 15.B3.19 map 94F-95A strain y: 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), NA0+ dependent acetaldehyde dehydrogenase (exaC), and pyrroloqulnoline qulnone synthesis A (pqqA) genes, complete cds; and pyrroloquinoline qulnone synthesis B (pqqB) gene.

partial cds.

Bacillus subtills wapA and orf genes for wall-associated protein and hypothetical proteins.

Bacillus subtilis genome containing the hut and wapA loci.

B.subtilis (Marburg 168) genes for beta-glucoside permease and betaglucosidase.

Home sapiens. SEQUENCING IN PROGRESS ,106 unordered pieces.

Homo sapens, SEQUENCING IN PROGRESS ~,106 unordered pieces.

Genomic sequence for Arabidopsis thaliana BAC F26F24, complete sequence.

Caenorhabdills elegans cosmid F07A1 1, complete sequence.

BNLGHi1 0201 Six-day Cotton fiber Gossyplum hirsutum cDNA 5' similar to (AC004684) hypothetical protein [Arabidopsls Ihaliana). mRNA sequence.

Caenorhabdlls elegans cosmid F07AI 1, complete sequence.

Streptomyces coelicotor Aeropyrum pernix Streptomyces coelicolor Mycobacteriumn tuberculosis Mycobacterium leprae Homo sapiens Drosophila melanogaster Drosophila melanogaster Rhodococcus erythropolis Mycobacterlum tuberculosis F'seudomonas aeruginosa Bacillus subtilis Bacillus subtills Bacillus subtilis Home sapiens Homo sapiens Arabidopsis thaliana Caenorhabditis elegans Gossyplumn hirsutumn 35.5 11 48,014 65,672 6 1,436 37,893 37,051 36,822 36,822 66,117 65,174 65,448 53,602 53,602 53.602 34,022 34,022 33,858 36,420 38,095 24-MAR-i1999 22-Jun-99 26-Apr-93 17-Jun-98 01-MAR-1 994 23-Nov-99 2-Aug-99 2-Aug-99 16-Jul-99 17-Jun-98 1 8-MAR-i1999 7-Feb-99 7-Feb-99 3-Jul-95 22-Aug-99 22-Aug-99 16-Apr-99 2-Sep-99 11-Jun-99 GBIN i:CEF07A1 1 35692 Z66511 Caenorhabditis elegans 33,707 2-Sep-99 2007203041 29 Jun 2007 rxa02572 668 GBBA1:MTCY63 GBBAI :MTCY63 GBHTGI:HS24H-I0 rxa02596 1326 GB-BA1:MTV026 GBBA2:AF026540 GBBA2:MTU96128 rxaO2611 1775 GBBAI:MTCYI3O GBBAi:MSGY151 GBBA1:U.00014 rxa02612 2316 GB-BA1:MTCYI3O GB-BA1 :MSGY1 51 GBBA1:STMGLGEN rxa02621 942 GB_8A1:CGL133719 GB-INI:CEM1O6 GBEST29:AiS47662 rxa02640 1650 GBBA1:MTVO25 GBBA1:PAU49666 38900 Z96800 38900 Z96800 46989 AL121632 23740 AL022076 1778 AF026540 1200 U96128 32514 Z73902 37036 AD000018 36470 U00014 32514 Z73902 37036 AD000018 2557 L11647 1839 AJ133719 39973 Z46935 377 A1547662 121125 AL022121 4495 U49666 1641 AB015974 512 N65787 65839 AC005916 88871 U58105 43411 AC004643 Table 4 (continued) Mycobactenumr tuberculosis H37Rv complete genome; segment 161 162. Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 161162. Mycobacteriumn tuberculosis H-omo sapiens chromosome 21 clone LLNI-116HO1124 map 21q21, Homo sapiens SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome; segment 157/1 62. Mycobacterium tuberculosis Mycobacterium tuberculosis UDP-galactopyranose mulase (gil) gone, complete Mycobacteriumn Mds. tuberculosis Mycobacterium tuberculosis UDP-galaclopyranose rnutase (gilo gene, complete Mycobacterium Wds. tuberculosis Mycobacterium tuberculosis H37Rv complete genome; segment 59/162. Mycobactedrn tuberculosis Mycobacteriumn tuberculosis sequence from clone y151. Mycobacteriumn tuberculosis Mycobacteriumn teprae cosmid B1549. Mycobacterium leprae Mycobacterium tuberculosis H37Rv complete genome; segment 5911162. Mycobacteriumn tuberculosis Mycobacterlumn tuberculosis sequence from clone yl 51. Mycobactenumr tuberculosis Streptomyces aureofaciens glycogen branching enzyme (g1gB) gene, complete Streptomyces cds. aureofaciens Corynebacterium glutamicumn yjcc gene. amtR gene and citE gene, partial. Corynebacteriumn glutamicum Caenorhabditis elegans cosmid M106, complete sequence. Caenorhabditis elegans UI-R-C3-sz-h-03-0-Ui.si UI-R-C3 Rattus norvegicus cDNA clone UI-R-C3-sz-h- Rattus norvegicus.

03-0-Ut mRNA sequence.

Mycobacterum tuberculosis H37Rv complete genome;, segment 155/162. Mycobacteriumn tuberculosis Pseudomonas aeruginosa (orIX), glycerol diffusion facilitator (gipF). glycerol Pseudomonas aeruginosa kinase (glpK). and Gip repressor (glpR) genes, complete cds, and (orfK) gene.

partial cds.

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

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

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

H-omo 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 57,486 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-98 17-Jun-98 29-Sep-99 24-Jun-99 30-OCT-i1998 25-MAR-1 998 17-Jun-98 10-DEC-1996 29-Sep-94 17-Jun-98 10-DEC-i1996 25-MAY-i1995 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 rxa02654 1008 GBBAI:AB015974 GBEST6:N65787 GBPL2:T17H3 GBRO:MMU58105 rxa02666 891 GBPR3:AC0046A3 2007203041 29 Jun 2007 GBPR3:ACOO.4643 43411 AC004643 GBBA2:AF049897 9196 AF049897 rxa02675 1980 GBOBA1:PDENQOURF 10425 L02354 GB-BA1:MTCY339 42861 Z77163 Table 4 (continued) H-omo sapiens chromosome 16, cosmid clone 363E3 (LANL), complete sequence.

Corynebacterium glutamicum N-acetylglulamnylphosphate reductase (argC).

ornithine acetyltransferase (argJ). N-acetylglutamale kinase (argB).

acetltornithtne transaminase (argD), ornithine cartbamoyltransferase (argF), arginine repressor (argR). argtninosuccinate synthase (argG). and argininosucclnate lyase (argH) genes. complete cds.

Paracoccus denitrificans NADH dehydrogenase (URF4). (NQ08). (NQO9). I (URF6), (NQOl0). (NQ01l1), (N0012). (NQ013). and (NQO1 4) genes, complete cds's- biotin (acetyl-CoA carboxyl] ligase (birA) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome: segment 101/1 62. 1 Myxococcus xanthus devR and devS genes, complete cdls's.

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

B.stearothermophilus Ict gene encoding L-tactate dehydrogenase. complete -omo sapiens Corynebacterium gIutamicum Paracoccus denitrificans vlycobacteriurn uberculosls vlyxococcus xanthus Bacillus caldolyticus Bacillus stearothermophilus Bacillus stearothermophilus )anio rerio )anio rerio 41.599 01-MAY-1998 40.413 1-Jul-98 40,735 20-MAY-1 993 36,471 17-Jun-98 GB_8A1:MXADEVRS 2452 rxa02694 1065 GB-BA1:BACLDH 1147 GB-BAI:BACLDHL 1361 GB-PAT:A06664 1350 rxa02729 844 GBEST15:AA494626 121 GB-EST1 S:AA494626 121 L19029 M1 9394 M14788 38,477 57,371 57.277 27-Jan-94 26-Apr-93 26-Apt-93 cds.

A06664 Bstearothefmophilus Ict gene.

AA494626 faO9dO4.r1 Zebrafish lCRFzfts Danio rerio cDNA clone 1 1A22 5 similar to TR:G 1171163 Gil171163 G/T-MISMATCH BINDING PROTEIN.;, mRNA sequence.

AiA494626 faO9dO4.rl Zebralish ICRFzfls Danio rerto cDNA dlone I 1A22 5 similar to TR:G1 171163 Gi 171163 GIT-MISMATCI1 BINDING PROTEIN.:;. mRNA sequence.

rxa02730 1161 GBESTI9:AA758660 233 GBEST15:AA494626 121 GB-PR4:AC006285 1501 rxa02737 1665 GB-PAT:E13655 2260 AA758660 ah67d06.sl Soares-testisN-T Homo sapiens cDNA clone 1320683 mRNA Homo sapiens sequence.

AA494626 faOgdO4.rl Zebrafish lCRFzfls Danio rerio cONA clone I 1A22 5 similar to Danio ferio, TR:G1 171163 Gil 171163 GrT-MISMATCH BINDING PROTEIN. mRNA seq uence.

AC006285 Homo sapiens, complete sequence. Homo sapiens El13655 gONA encoding glucose-6-phosphate dehydrogenase. Corynebacteriu 57,277 29-Jul-93 50.746 27-Jun-97 i 36,364 27-Jun-97 37.059 29-DEC-i1998 42,149 27-Jun-97 37,655 15-Nov-99 99.580 24-Jun-98 38,363 19-Jun-98 172 m GB-BA:MTCY493 40790 Z95844 Mycobacterium tuberculosis H37Rv complete genome; segment 631162.

GBBA1:SCSA7 rxa02738 1203 GBPAT:E13655 GBBA1:SCC22 GBBA1:SC5A7 rxa02739 2223 GBBA1:AB023377 40337 AL031 107 Streptomyces coelicolor cosmid 5A7.

2260 E13655 gDNA encoding glucose-6-phosphate dehydrogenase.

glutamicum Mycobacterium tuberculosis Streptomyces coelicolor Corynebacterium glutamicum Streptomyces coelicolor Streptomyces coelicolor Corynebacterium 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 AB023377 Streptomyces coeticolor cosmid C22.

Streptomyces coelicolor cosmid 5A7.

Corynebacterium glutamicum tkt gene for Iransketolase. complete cds.

2007203041 29 Jun 2007 GBBA1:MLCL536 GBBA1:U00013 rxa02740 1053 GBHTG2:AC006247 GBHTG2:AC006247 GBHTG3:AC007150 rxa02741 1089 GB_-TG2:AC004951 GBHTG2:AC004951 GB iNl:A03006546 rxa02743 1161 GBOAI:MLCL536 GBBAI:U00013 GBHTG2:AC007401 rxa02797 1026 GBBA1:CGBETPGEN GBGSS9:AQ148714 GBBA1:BFU64514 Table 4 (continued) 36224 Z99125 Mycobacterlum Ieprae COSMld L536. Mycobacterium, leprae 35881 U00013 Mycobacterium leprae cosmid 61496. Mycobacterium teprae 174368 AC006247 Drosophila melanogasler chromosome 2 clone BACR481 10 (D50S) RPCI-98 Drosophila melanogas 48.1.10 map 49E&-49F8 strain y; cn bw sp, SEQUENCING IN PROGRESS -,17 unordered pieces.

174368 AC006247 Drosophila melanogaster chromosome 2 clone BACR48I110 (D505) RPCI-98 Drosophila melanogas 48.1.10 map 49E6-49F8 strain y; cn bw sp, SEQUENCING IN PROGRESS -,17 unordered pieces.

121474 AC007150 Drosophila melanogaster chromosome 2 clone BACRI6PI3 (0597) RPCI-98 Drosophila melanogas 16.P.13 map 49E-49F strain y; cn bw sp, -SEQUENCING IN PROGRESS-, 87 unordered pieces.

129429 AC004951 Homo sapiens clone D.11022114, -SEQUENCING IN PROGRESS 14 Homo sapiens unordered pieces.

129429 AC004951 Homo sapiens clone W1022114, -SEQUENCING IN PROGRESS- 14 Homo sapiens unordered pieces.

931 AB0065-46 Ephydatla fluvlatilis mRNA for G protein a subunit 4, partial cds. Ephydatia fluv'iatilis 36224 Z99125 Mycobacterium leprae cosmid L536. Mycobacterium Ieprae 35881 U00013 Mycobacterlum leprae cosmid B1496. Mycobacterium Ieprae 83657 AC007401 Homo sapiens clone NHOSO51007. SEQUENCING IN PROGRESS 3 Homo sapiens unordered pieces.

2339 X93514 C.glutamlcum betP gene. Corynebacterium glutamicum 405 AQ148714 HS_3136_AlA03_MR CIT Approved Human Genomic Spermn Library D Homo Homo sapiens sapiens genomic clone Plate=3136 CoI=5 Row=A, genomic survey sequence.

3837 U64514 Bacillus firmus dppABC operon. dipepllde transporter protein dppA gene, Bacillus lirmus partial cds, and dipeptide transporter proteins dppB and dppC genes, complete cds.

36947 U00020 Mycobacterium Ieprae casmld B229. Mvcobacterium lenrae ter ler 37.105 ter 38,728 33,116 33,116 36,379 48,401 48,401 37.128 38,889 34.321 61,573 61,573 37,105 04-DEC. 1998 01-MAR.1994 2-Aug.99 2-Aug-99 2 0-Sep-99 12-Jun-98 12-Jun-98 23-Jun-99 04-DEC.1998 01-MAR.1994 26-Jun-99 8-Sep-97 08-OCT-I1998 38,072 1-Feb-97 rxa02803 680 rxa02821 363 GDBA1:U00020 GBBA2:PSUB5643 GBBA1:5C6G4 GBHTG2:AC008105 GBHTG2:AC008105 34,462 4032 41055 91421 91421 U85643 AL03 1317 AC008 105 AC0081 05 Pseudomonas syrlngae pv. syringae putative dihydropleroate synthase gene, Pseudomonas syringae pv. 50,445 partial cds, regulatory protein MrsA (mrsA), triose phosphate Isomerase (IpIA), syringae transport protein SecG (secG), tRNA-Leu. tRNA-Met, and 15 kDa protein genes, complete cds.

Streptomyces coelicolor cosmid 6G4. Streptomyces coelicclor 59,314 H-omo sapiens chromosome 17 clone 2020_K_17 map 17, SEQUENCING Homo sapiens 37,607 IN PROGRESS"'~, 12 unordered pieces.

Homo sapiens chromosome 17 clone 2020_K_17 map 17, SEQUENCING Homo sapiens 37.607 IN PROGRESS 12 unordered pieces.

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

01 -MAR-i1994 9-Apr-97 20-Aug.98 22-Jul-99 22-Jul-99 30-Jun-99 GBE5T33:AV117143 222 AV 117143 2007203041 29 Jun 2007 rxa02829 373 GBHTG1:HSUgG8 GBHrG1:HSU9G8 GB-PR3:HSU8585 rxc-03216 1141 GB-HTG3:AC008184 48735 48735 39550 151720 AL008714 AL008714 Z69724 AC008 184 GB-EST15:AA477537 411 AA477537 Table 4 (continued) Homo sapiens chromosome X clone LLOXNCO1-9G38, SEQUENCING IN Homo sapiens PROGRESS in unordered pieces.

Homo sapiens chromosome X clone LLOXNCO1-9G8. -SEQUENCING IN Homo sapiens PROGRESS in unordered pieces.

Human DNA sequence from cosmid U85B5. between markers DXS366 and .Homo sapiens DXS87 on chromosome X.

Drosophila melanogaster chromosome 2 clone 63ACRO4005 (D540) RPCI-98 Drosophila melanogaster 04.13.5 map 36E5-36F2 strain y; cn bw sp. SEQUENCING IN PROGRESS 27 unordered pieces.

zu36g12.rl Soares ovary tumor NbHOT H-omo sapiens cDNA clone Homo sapiens IMAGE:740134 5'similar to contains Alu repetitive elemenl~conlalns element HGR repetitive element;. mRNA sequence.

fa9ldOB.yl zebrafish fin dayl regeneration Danio rerio cDNA mRNA Danio rerlo sequence.

Streptomyces coelicolor cosmid 3F9. Streptomyces coellcolor A3(2) Slincolnensis (78-11) Lincomycin production genes. Streptomyces lincolnensis Homo sapiens chromosome 15 clone RP1 1-424J110 map 15, SEQUENCING Homo sapiens IN PROGRESS 41 unordered pieces.

Homo sapiens chromosome 19, cosmid R302 17, complete sequence. Homo sapiens Spombe chromosome I cosmld c926. Schizosaccharomyces pombe Archaeoglobus fulgidus section 26 of 17201f the complete genome. Archaeoglobus fulgidus 41.595 41,595 41.595 39.600 23-Nov-99 23-Nov-99 23-Nov-99 2-Aug-99 37.260 9-Nov-97 GB-EST26:A1330662 rxs03215 1038 GBBA1:5C3F9 GBBA1:SLLINC GBHTG5:AC009660 rxs03224 1288 GBPR3:AC004076 GBPL2:5PAC926 GB-BA2:AEOOI 081 412 19830 36270 204320 41322 23193 11473 A1330662 AL023862 X79146 AC009660 AC004076 AL1 10469 AE001081 37,805 48.657 39.430 35.15 1 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 15-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°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/1 MgSO, x 7H,0, 10 ml/I KH 2 PO, solution (100 g/1, adjusted to pH 6.7 with KOH), 50 ml/l M12 concentrate (10 g/1 (NI-) 2 SO,, 1 g/l NaCI, 2 g/1 MgSO, x 0.2 g/1 CaCI,, 0.5 g/l yeast extract (Difco), 10 ml/I trace-elements-mix (200 mg/1 FeSO, x H 2 0, 10 mg/l ZnSO, x 7 HO, 3 mg/1 MnCI, x 4 H 2 0, 30 mg/l HBO, 20 mg/l CoC1, x 6 HO, 1 mg/l NiCI, x 6 H,0, 3 mg/1 NaMoO, x 2 HO, 500 mg/1 complexing agent (EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/l biotin, 0.2 mg/l folic acid, mg/l p-amino benzoic acid, 20 mg/l riboflavin, 40 mg/l ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/l myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37C, 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 jpg/ml, the suspension is incubated for ca. 18 h at 37°C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroformisoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20°C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing pg/ml RNaseA and di.alysed at 4°C against 1000 ml TE-buffer for at least 3 hours.

During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added. After a min incubation at -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 pHM 1519 or pBL1) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley Sons) to which a origin or replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E.

coli and C. glutamicum, and which can be used for several purposes, including gene over-.

expression (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 shuftle vectors described above and to introduce such a hybrid vectors into strains of Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984)J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described e.g. in Schifer, 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 pCGI Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, and A. J.

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

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

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

-122- (1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information is evidence of the degree of transcription of the mutant gene. Total cellular RNA can be prepared from Corynebacteriumglutamicum by several methods, all well-known in the art, such as that described in Bormann, E.R. et al.

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

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

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

Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be 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 NIHCI 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 I (Merck) or BHI (grain heart infusion, DIFCO) or others.

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

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

An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It 124 is also possible to maintain a constant culture pH through the addition of NaOH or NH.OH 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 oo of 0.5 1.5 using cells grown on agar plates, such as CM plates (10 g/l glucose, 2,5 g/1 NaC1, 2 g/1 urea, 10 g/l 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/1 meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30'C. Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.

Example 8 -In vitro Analysis of the Function of Mutant Proteins The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one of ordinary skill in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be 125- N found, for example, in the following references: Dixon, and Webb, (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.

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

Press: Oxford; Boyer, ed. (1983) The Enzymes, 3 r d ed. Academic Press: New j- York; Bisswanger, (1994) Enzymkinetik, 2 nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, Bergmeyer, GraI3, 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- SUllman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: g 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.

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

O (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, Ce¢ O 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 supematant 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-

O

S90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST a programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST n- nucleotide searches can be performed with the NBLAST program, score 100, C^ 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 Shomologous to SMP protein molecules of the invention. To obtain gapped alignments Cc€ Sfor 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 129- Swere compared to genes present in Genbank in a three-step process. In a first step, a g 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 C 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 O subsequently globally aligned to each of the top three FASTA hits, using the GAP Sprogram in the GCG software package (using standard parameters). In order to obtain 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 nf A-Z 1_ 130- C< may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or-absolute amount of all or selected nucleic acids in the applied nucleic C 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 which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270: 467-470).

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

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

Hybridization of labeled nucleic acids to microarrays is described in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. (1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for example, as -131- NC 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 CI 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 O for specific and/or desired strain properties such as pathogenicity, productivity and Sstress 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 3 SS-methionine, 35S-cysteine, "C-labelled amino acids, 1 SN-amino acids, "N0 3 or "NH4 or 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, amongothers). 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:85, or a complement thereof.

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:86, 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:86, 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:85, 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:85, 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:86, 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:86.

17. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:86.

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

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:86. An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:86, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:86. 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

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

30. The method of claim 29, wherein said amino acid is selected from the ;Z group consisting of: lysine, glutamate, glutamine, alanine, aspartate, glycine, c 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 Ci cell whose genomic DNA has been altered by the introduction of a nucleic acid Smolecule 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:85, wherein the nucleic acid molecule is disrupted.

34. A host cell comprising the nucleic acid molecule of SEQ ID NO:85, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID A host cell comprising the nucleic acid molecule of SEQ ID NO:85, 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 P20679AU05 Associated Physical Media Submitted EI~ Basic Document (ie Conventi 1111 Verified Translation LII] Description [II] Claims [II] Abstract [III]Drawings [III]Gene Sequence Listing [I]CD-Rom 4Diskette Other Eli] (eg. Deeds, Assignrr ion/Priority Document) ents, etc.)