AU2007203042A1 - Corynebacterium glutamicum genese encoding proteins involved in carbon metabolism and energy production - Google Patents

Description

P001 Section 29 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Corynebacterium glutamicum 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: COR YNEBACTERIUM GLUTAMICUM GENES ENCODING PROTEINS INVOLVED IN CARBON METABOLISM AND ENERGY PRODUCTION Related Applications This application claims priority to prior U.S. Provisional Patent Application Serial No. 60/141031, filed June 25, 1999, U.S. Provisional Patent Application Serial No. 60/143208, filed July 9, 1999, and U.S. Provisional Patent Application Serial No.

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

19931413.6, filed July 8, 1999, German Patent Application No. 19931419.5, filed July 8, 1999, German Patent Application No.. 19931420.9, filed July 8, 1999, German Patent Application No. 19931424. 1, filed July 8, 1999,. German Patent Application No.

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

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

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

19933005.0, filed July 14, 1999, German Patent Application No. 19940765.7, filed August 27, 1999, German Patent Appication 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. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as sugar metabolism and oxidative phosphorylation (SMP) proteins.

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

The SMP nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. gluramicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.

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

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

The SMP proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et 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 e 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 ;Z 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 C, 5 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) Sand the reducing equivalents (such as NADH or NADPH) produced by these metabolic 10 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:245, or a complement thereof.

An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:246, 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:246, 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:245, or a complement thereof.

An isolated nucleic acid molecule comprising a fragment of at least Scontiguous nucleotides of the nucleotide sequence of SEQ ID NO:245, or a Scomplement thereof.

c* An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino Sacid sequence of SEQ ID NO:246, or a complement thereof.

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

An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:246.

An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:246.

An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:245.

An isolated polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:246.

An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:246, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:246.

An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:245.

A host cell comprising the nucleic acid molecule of SEQ ID NO:245, wherein the nucleic acid molecule is disrupted.

A host cell comprising the nucleic acid molecule of SEQ ID NO:245, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:245.

A host cell comprising the nucleic acid molecule of SEQ ID NO:245, 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.

0 In another embodiment, the isolated nucleic acid molecule is at least nucleotides in length and hybridizes under stringent conditions to a nucleic acid Smolecule comprising a nucleotide sequence of the invention a sequence of an odd- Snumbered 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 sequences 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. gluatmicum 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 -11- O impact which nonetheless results in an increase of yield, production, and/or efficiency of Sproduction 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 r d 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 productionand 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 ofaketoglutarate, 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 of aspartate. Isoleucine is formed from threonine. A complex 9-step pathway results in the production ofhistidine 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 r d ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in terms of energy, precursor molecules, and the enzymes necessary to synthesize them.

Thus it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own 14production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3 rd ed. Ch. 24: "Biosynthesis of Amino Acids and Heme" p.

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

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

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

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

Thiamin (vitamin B 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 offlavin 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 B 1 2 is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.

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

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

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

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

Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in 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, ca-1,l linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et (1998) U.S.

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

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

(1980) Industrielle Mikrobiologie, Springer: Berlin; Bartholomew, and Reiman, H.B. (1979). Economics of Fermentation Processes, in: Peppier, H.J. and Perlman, D., eds. Microbial Technology 2 n d 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 CO 2 The acetyl group of acetyl CoA then reacts with -21 the 4 carbon unit, oxaolacetate, leading to the formation of citric acid, a 6 carbon organic acid. Dehydration and two additional CO 2 molecules are released. Ultimately, 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.

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

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

Non-hexose carbon substrates may also serve as carbon and energy sources for cells. Such substrates may first be converted to hexose sugars in the gluconeogenesis pathway, where glucose is first synthesized by the cell and then is degraded to produce energy. The starting material for this reaction is phosphoenolpyruvate (PEP), which is one of the key intermediates in the glycolytic pathway. PEP may be formed from substrates other than sugars, such as acetic acid, or by decarboxylation of oxaloacetate (itself an intermediate in the TCA cycle). By reversing the glycolytic pathway (utilizing a cascade of enzymes different than those of the original glycolysis pathway), glucose-6phosphate may be formed. The conversion ofpyruvate to glucose requires the utilization of 6 high energy phosphate bonds, whereas glycolysis only produces 2 ATP in the conversion of glucose to pyruvate. However, the complete oxidation of glucose (glycolysis, conversion ofpyruvate 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, -23the 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 glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032. The nucleotide sequence of the isolated C. glutamicum SMP DNAs and the predicted amino acid sequences of the C.

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

The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention -26the sequence of an even-numbered SEQ ID NO of the Sequence Listing). As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.

An SMP protein or a biologically active portion or fragment thereof of the invention can participate in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or can have one or more of the activities set forth in Table 1.

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

The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid sequences located at the 5' and 3' ends of the nucleic acid) in the -27genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated SMP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived a C. glutamicum cell). Moreover, an "isolated" nucleic acid molecule, such as a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a C. glutamicum 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 -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 glulamicum.

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

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

Portions of proteins encoded by the SMP nucleic acid molecules of the invention are preferably biologically active portions of one of the SMP proteins. As used herein, the term "biologically active portion of an SMP protein" is intended to include a portion, a domain/motif, of an SMP protein that participates in the metabolism of carbon -32compounds such as sugars, or in energy-generating pathways in C. glutamicum, or has an activity as set forth in Table 1. To determine whether an SMP protein or a biologically active portion thereof can participate in the metabolism of carbon compounds or in the production of energy-rich molecules in C. glutamicum, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary skill in the art, as detailed in Example 8 of the Exemplification.

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

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

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

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

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

Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. 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 -34 another embodiment, an isolated nucleic acid molecule of the invention is at least nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of of an odd-numbered SEQ ID NO: of the Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley Sons, N.Y. (1989), 6.3.1-6.3.6.

A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0. 1% SDS at 50-65 0 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleotide sequence of the invention corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C.

glutamicum SMP protein.

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

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding SMP proteins that contain changes in amino acid residues that are not essential for SMP activity. Such SMP proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the SMP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of the invention and is capable of participate in the metabolism of carbon compounds such as sugars, or in the biosynthesis of high-energy compounds in C. gluramicum, 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 I 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- -38galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, acid wybutoxosine, pseudouracil, queosine, 2-thiocytosine, methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid 5-methyl-2-thiouracil, 3-(3-amino-3-N-2carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

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

In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual p-units, the strands run parallel to each other (Gaultier 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-, Ipp-, lac-, Ipp-lac-, lacI q T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SP02, 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 tumefaciens -mediated transformation of Arabidopsis 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, pHSI, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- III 113-B 1, Xgtl 1, pBdCl, and pET 1 Id (Studier et al., Gene Expression Technology.

Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).

Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS 174(DE3) from a resident X prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJ01, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds.

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

-43- One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the SMP protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), 2 t, pAG-1, Yep6, Yep 13, 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+, -44pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.

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

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

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

O a manner which allows for expression (by transcription of the DNA molecule) of an SRNA molecule which is antisense to SMP mRNA. Regulatory sequences operatively ;Z linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.

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

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

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

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid linear DNA or RNA a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector a plasmid, phage, phasmid, phagemid, -46transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.

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

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

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

In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous SMP gene is functionally disrupted no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous SMP gene is mutated or otherwise altered but still encodes functional protein the upstream regulatory region can be altered to thereby alter the expression of the endogenous SMP protein). In the homologous recombination vector, the altered portion of the SMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the SMP gene to allow for homologous recombination to occur between the exogenous SMP gene carried by the vector and an endogenous SMP gene in a microorganism. The additional 47flanking SMP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see Thomas, and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism by electroporation) and cells in which the introduced SMP gene has homologously recombined with the endogenous SMP gene are selected, using art-known techniques.

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

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

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

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce express) an SMP protein. Accordingly, the invention further provides methods for producing SMP proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an SMP protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered SMP protein) in a suitable medium until SMP protein is produced. In another -48- Sembodiment, the method further comprises isolating SMP proteins from the medium or .s the host cell.

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

-49- An isolated SMP protein or a portion thereof of the invention can participate in the metabolism of carbon compounds such as sugars, or in the production of energy 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 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 is preferably a biologically active portion as described herein. In another preferred embodiment, an SMP protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing. In yet another preferred embodiment, the SMP protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, hybridizes under stringent conditions, to a nucleotide sequence of the invention a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing). In still another preferred embodiment, the SMP protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to one of the nucleic acid sequences of the invention, or a portion thereof. Ranges and identity values intermediate to the above-recited values, 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. The preferred SMP proteins of the present invention also preferably possess at least one of the SMP activities described herein. For example, a preferred SMP protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or which has one or more of the activities set forth in Table 1.

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

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

Typically, biologically active portions (peptides, peptides which are, for example, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of an SMP protein. Moreover, other -51 biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of an SMP protein include one or more selected domains/motifs or portions thereof having biological activity.

SMP proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the SMP protein is expressed in the host cell. The SMP protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, an SMP protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native SMP protein can be isolated from cells endothelial cells), for example using an anti-SMP antibody, which can be produced by standard techniques utilizing an SMP protein or fragment thereof of this invention.

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

-52- SPreferably, 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 C 5 termini for ligation, restriction enzyme digestion to provide for appropriate termini, 0 filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid Ce¢ 0undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.

0Alternatively, 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 Sl nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the SMP protein.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of SMP homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the -54frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify SMP homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

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

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

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

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

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

The SMP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and energy-releasing processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the -56evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.

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

The invention provides methods for screening molecules which modulate the activity of an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more SMP proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the SMP protein is assessed.

There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. gluramicum 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 1 -58due to the presence of a greater number of viable cells, each producing the desired fine chemical.

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

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

Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and protein molecules of the invention may be utilized to generate C. glutamicum 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.

2007203042 29 Jun 2007 TABLE 1: GENES IN THE APPLICATION

HMP:

Nucleic Acid SEQ ID NO 3 7

TCA:

Nucleic Acid SEQ ID NO 9 11 13 17 19 21 Amino Acid SEQ ID NO 2 4 6 8 Amino Acid SEQ I0 NO 10 12 14 16 18 20 22 Identification Code RXS02735 RXA01626 RXA02245 RXA01 015 Identification Code RXNO013 12 F RXA01312 RXN00231 RXA01311 RXA01 535 RXA0051 7 RXA01 350 Identific-ation Code RXA02 149 RXA01814 RXN02803 F RXA02803 RXN03076 F RXA02854 RXA00511 CLOnfg WV0074 GR00452 GROO654 GR00290 WV0082 GR00380 WV0083 GR00380 GR00427 GROO131 GR00392 GRO0639 GROO515 WV0086 GR00784 WV0043 GR1 0002 GROC 129 NT Start NT Stop Function 14576 4270 13639 346 15280 3926 14295 5 6-Phosphoglucolactonase L-ribulose-phosphate 4-epimerase RIBULOSE-PHOSPH-ATE 3-EPIMERASE (EC 5.1.3. 1) RIBOSE 5-PHOSPHATE ISOMERASE (EC 5.3.1.6) NT Start NT Stop Function 20803 2690 15484 1611 1354 1407 1844 18785 1614 14015 865 2760 2447 2827 SUCCINATE DEHYDROGENASE FLAVOPROTEIN SUBUNIT (EC 1.3.99.1) SUCCINATE DEHYDROGENASE FLAVOPROTEIN SUBUNIT (EC 90.1) SUCCINATE-SEMIALIDEHYDE DEHYDROGENASE (NADP+) (EC 1.2.1.16) SUCCINATE DEHYDROGENASE IRON-SULFUR PROTEIN (EC 1.3.99.1) FUMARATE H-YDRATASE PRECURSOR (EC 4.2.1.2) MALATE DEHYDROGENASE (EC 1.1.1.37) (EC 1.1.1.82) MALATE DEHYDROGENASE (EC 1.1.1.37) EMB-Pathway Nucleic Amino Acid Acid SEQ SEQ ID NO ID NO 23 24 26 27 28 29 30 31 32 33 34 36 NT Start NT Stop Function 17786 2571 2 1624 1588 18754 910 657 400 35 5 513 GLUCOKINASE (EC 2.7.1.2) PHOSPHOGLUCOMUTASE (EC 5.4.2.2)1/ PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) PI-OSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHOSPHOGLUCOMUTASE (EC 5.4.2.2)1/ PHOSPHOMANNOMUTASE (EC 5.4.2.8) PHO0SPHOGLUCOMUTASE (EC 5.4.2.2)1/ PHOSPHOMANNOMUTASE (EC 5.4.2.8) 2007203042 29 Jun 2007 Table I (continued) Nucleic Acid Amino Acid Identification Code Cni. NT Start NT Stop Function SEQ ID NO SEQ IDNO 37 38 RXN01 365 WV0091 1476 103 PHOSPHOGLUCOMUTASE (EC 5.4.2.2) PHOSPHOMANNOMUTASE (EC 5.4.2.8) 39 40 F RXA0 1365 GR00397 897 4 PH-OSPHOGLUCOMUTASE (EC 5.4.2.2)1 PHOSPHOMANNOMUTASE (EC 5.4.2.8) 41 42 RXA00098 GROO014 6525 8144 GLUCOSE-6-PHOSPHATE ISOMERASE (GPI) (EC 5.3.1.9) 43 44 RXA0 1989 GR00578 1 630 GLUCOSE-6-PHOSPHATE ISOMERASE A (GPI A) (EC 5.3.1.9) 46 RXAO34 GR00059 1549 2694 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 47 48 RXA02492 GRG0720 2201 2917 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 49 50 RXA06381 GR00082 1451 846 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 51 52 RXA02 122 GR00636 6511 5813 PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) 53 54 RXA00206 GR00032 6171 5134 6-PHOSPHOFRUCTOKINASE (EC 2.7.1.11) 56 RXA01243 GR00359 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 GR00479 1397 366 FRUCTOSE-BISPHOSPHATE ALDOLASE (EC 4.1.2.13) 61 62 RXA02258 GR00654 26451 27227 TRIOSEPHOSPHATE ISOMERASE (EC 5.3.1.1) 63 64 RXN01225 WV0064 6382 4943 GLYCERALt3EHYDE 3-PHOSPHATE DEHYOROGENASE (EC 1.2.1.12) 66 F RXA01225 GR00354 5302 6741 GLYCERALDEHYD E-3-PH-OSP HATE IDEHYDROGENASE HOMOLOG 67 68 RXA02256 GR00654 23934 24935 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (EC 1.2.1.12) 69 70 RXA02257 GR00654 25155 26369 PHOSPHOGLYCERATE 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 KtNASE (EC 2.7.1.40) 76 RXN02675 WV0098 72801 70945 PYRUVATE KINASE (EC 2.7.1.40) 77 78 F RXA02675 GR00754 2 364 PYRUVATE KINASE (EC 2.7.1.40) C 79 80 F RXA02695 GR00755 2949 4370 PYRUVATE KINASE (EC 2.7.1.40) 81 82 RXA00682 GR00179 5299 3401 PHOSPHOENOLPYRUVATE SYNTHASE (EC 2.7.9.2) 83 84 RXA00683 GR00179 6440 5349 PHOSPHOENOLPYRUVATE SYNTHASE (EC 2.7.9.2) 86 RXN00635 WV0135 22708 20972 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC 1.2.2.2) 87 88 F RXA02807 GR00788 88 552 PYRUVATE DEHYDROGENASE (CYTrOCHROME) (EC 1.2.2.2) 89 90 F RXA00635 GR00167 3 923 PYRUVATE DEHYDROGENASE (CYTOCHROME) (EC 1.2.2.2) 91 92 RXN03044 WV0019 1391 2221 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 93 94 F RXA02852 GR00852 3 281 PYRUVATE DEH-YDROGENASE El COMPONENT (EC 1.2.4.1) 96 F RXA00268 GROO041 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 GR10022 411 4 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 101 102 RXN03043 Woo019 1 1362 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 103 104 F RXA02897 GR10039 1291 5 PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) 105 106 RXN03083 WV0047 88 1110 DIHYDROUIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 107 108 F RXA02853 GRIO001 89 1495 DIHYDROLIPOAMIDE DEHYDROGENASE (EC 1.8.1.4) 109 110 RXA02259 GR00654 27401 30172 PHOSPHOENOLPYRUVATE CARBOXYLASE (EC 4.1.1.31) Ill 112 RXN02326 WV0047 4500 5315 PYRUVATE CARBOXYLASE (EC 6.4. 1.1) 113 114 F RXA02326 GR00668 5338 4523 PYRUVATE CARBOXYLASE 115 116 RXN02327 WV0047 3533 4492 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 117 118 F RXA02327 GR00668 6305 5346 PYRUVATE CARBOXYLASE 119 120 RXN02328 WV0047 1842 3437 PYRUVATE CARBOXYLASE (EC 6.4. 1. 1) 121 122 F RXA02328 GR00568 7783 6401 PYRUVATE CARBOXYLASE (EC 6.4.1.1) 123 124 RXN01048 VV0079 12539 11316 MALtC ENZYME (EC 1.1.1.39) 2007203042 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 125 127 129 131 133 135 137 139 141 143 145 147 149 151 153 155 Amino Acid SEQ ID NO 126 128 130 132 134 136 138 140 142 144 148 148 150 152 154 156 Identification Code F RXA01048 F RXA00290 RXA02694 RXN00296 F RXA00296 RXA01901 RXN01 952 F RXA01952 F RXA01955 RXA00293 RXN01 130 F RXA01I130 RXN031 12 F RXA01 133 RXN00871 F RXA00871 RXN02829 F RXA02829 RXN0 1468 F RXA01468.

RXA00794 RXN02920 F RXA02379 RXN02688 RXN03087 RXN03186 RXN03187 RXN02591 RXS01 260 RXS01 261 GR00296 GR00046 GRD0755 WV0176 GR00048 GRO0544 WO0105 GR00562 GR00562 GR00047 WO0157 GRD0315 W085 GR0O3 16 W0127 GRD0239 W0354 GR00816 W019 GRO0422 GROO211 W02 13 GRD0690 W0098 W0052 WV0377 W0382 W0098 W0009 W0009 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) 1-LACTATE DEHYDROGENASE (EC 1.1.1.27) 0-LACTATE DEHYOROGENASE (CYTOCHROME) (EC 1.1.2.4) 0-LACTATE DEHYDROGENASE (CYTOCHROME) (EC 1.1.2.4) 1-LACTATE DEHYDROGENASE (CYTOCHROME) (EC 1.1.2.3) D-LACTATE DEHYDROGENASE (EC 1.1.1.28) 0-LACTATE DEHYDROGENASE (EC 1.1.1.28) 0-LACTATE DEHYDROGENASE (EC 1.1.1.28) 0-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1.1.1.95) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1. 1. 1.95) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1. 1. 1.95) D-3-PH-OSPHOGLYCERATE DEHYbROGENASE (EC 1. 1. 1.95) IOLB PROTEIN I016 PROTEIN: 0-FRUCTOSE 1,6-BISPHOSPHATE GLYCERONE-CC PHOSPHATE 0- GLYCERALDEHYDE 3-PHOSPHATE.

IOLS PROTEIN IOLS PROTEIN NAG0 PROTEIN PUTATIVE N-GLYCERALDEHYDE-2-PHOSPHOTRANSFERASE GLPX PROTEIN D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1. 1. 1.95) D-3-PHOSPHOGLYCERATE DEHYDROGENASE (EC 1. 1. 1.95) PHOSPHOGLYCERATE MUTASE (EC 5.4.2.1) PYRUVATE CARBOXYLASE (EC 6.4.1.1) PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) PYRUVATE DEHYDROGENASE El COMPONENT (EC 1.2.4.1) PHOSPHOENOLPYRUVATE CARBOXYKINASE (GTPJ (EC 4.1.1.32) LIPOAMIDE DEHYDROGENASE COMPONENT (E3) OF BRANCHED- CHAIN 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 Identification Code SEQ ID NO 186 RXA02640 188 RXN01025 190 F RXA0I1025 192 RXA01851 194 RX.A01242 196 RXA02288 Contig GR00749 W01 43 GR00293 GR00525 GR00359 GRO0661 NT Start NT Stop Function 2926 4488 1853 1830 2302 147 GLYCEROL KINASE (EC 2.7.1.30) GLYCEROL-3-PHOSPHATE DEHYDROGENASE (EC 1.1.1.94) GLYCEROL-3-PHOSPHATE DEHYDROGENASE (EC 1.1.1.94) AEROBIC GLYCEROL-3-PHOSPHATE DEHYDROGENASE (EC 1.1.99.5) GLYCEROL-3-PHOSPHATE REGULON REPRESSOR GLYCEROL-3-PHOSPHATE REGULON REPRESSOR 2007203042 29 Jun 2007 Table I (continued) Nucleic Acid Amino Acid Identification Code Contig. NT Start NT Stop Function SEQ ID NO SEQ ID NO 197 198 RXN01691 199 200 201 202 203 204 F RXA01891 RXA02414 RXN01 580 WV0122 24949 24086 GLYCEROL-3-PHOSPH-ATE-BINDING PERIPLASMIC PROTEIN

PRECURSOR

GR0054 1 1736 918 GLYCEROL-3-PHOSPHATE-BINDING PERIPLASMIC PROTEIN

PRECURSOR

GR00703 3808 3062 Uncharacterized protein involved in glycerol metabolism (homolog of Drosophila rhomboid) WV0122 22091 22807 Glycerophosphoryl diester phosphodiesterase 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 RXAO15711 213 214 RXA01572 215 216 RXA01758 217 218 RXA02539 219 220 RXN03061 221 222 RXN03150 223 224 RXN01 340 225 226 RXN01498 227 228 RXN02674 229 230 RXN00868 231 232 RXN01 143 233 234 RXNO 1146 235 236 RXN01 144 ontig NT Start NT Stop Function GROO.41 8 2547 1357 ACETATE KINASE (EC 2.7.2.1) GR00179 8744 7941 ACETATE OPERON REPRESSOR GRO0037 4425 3391 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) GR00438 1360 1959 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) GR00438 1928 2419 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) GR00498 3961 2945 ALCOHOL DEHYDROGENASE (EC 1.1.1.1) GR00726 11676 10159 ALDEHYDE DEHYDROGENASE (EC W0034 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) W0008 1598 3160 ALDEHYDE DEHYDROGENASE (EC 1.2.1.3) W0315 15614 14163 ALDEH-YDE DEHYDROGENASE (EC 1.2.1.3) W0127 2230 320 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) W0077 9372 8254 ACETOLACTATE SYNTHASE LARGE SUBUNIT (EC 4.1.3.18) W0264 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 Coh.

SEQ ID NO SEQ ID NO 237 238 RXA02474 GRO07 239 240 RXA02453 GRO07 241 242 RXS01758 W01Ii NT Start NT Stop Function 15 8082 7309 (S,S)-butane-2,3-dioldcehydrogenase (EC 1. 1. 1.76) 10 6103 5351 ACETOIN(DIACETYL) REDUCTASE (EC 1.1.1.5) 2 27383 28399 ALCOHOL DEHYDROGENASE (EC 1. 1.1. 1) 2007203042 29 Jun 2007 Table I (continued) HMP-Cycle Nucleic Acid SEQ ID NO 243 245 247 249 Amino Acid Identification Code SEQ 10 NO 244 RXA02737 246 RXA02738 248 RXA02739 250 RXA00965 Contig NT Start NT Stop Function GR00763 3312 1771 GLUCOSE-6-PHOSPHATE 1-DEHYOROGENASE (EC 1.1.1.49) GR00763 4499 3420 TRANSALDOLASE (EC 2.2.1.2) GR00763 6769 4670 TRANSKETOLASE (EC 2.2.1.1) GR00270 1232 510 6-PHOSPHOGLUCONATE DEHYDROGENASE, OECARBOXYLATING (EC 1.1.1.44) W0106 2817 1366 6-PHOSPHOGLUCONATE DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44) GR00283 3012 4448 6-PHOSPHOGLUCONATE DEHYDROGENASE, DECARBOXYLATING (EC 1.1.1.44) 251 252 253 254 RXN00999 F RXA00999 Nucleotide sugar conversion Nucleic Acid Amino Acid SEQ ID NO SEQ ID NO Identification Code RXN02596 F RXA02596 F RXA02642 RXA02572 RXA02485 RXA0 1216 RXA0 1259 RXA02028 RXAO1 262 RXA01 377 RXA02063 RXN00014 F RXAOD0 14 RXAO 1570 RXA02666 RXA00825 Contie.

VV0098 GR00742 GR00749 GR00737 GR00718 GR00352 GR00367 GR00616 GR00367 GROO400 GR00626 W0048 GROO002 GR00438 GR00753 GR00222 NT Start NT Stop Function 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 1261 6493 1154 UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UDP-GALACTOPYRANOSE MUTASE (EC 5.4.99.9) UDP-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-l -PHOSPHATE GUANYLTRANSFERASE (EC 2.7.7.13) GLUCOSE-I -PHOSPHATE ADENYLYLTRANSFERASE (EC 2.7.7.27) GLUCOSE-I -PHOSPHATE TI-YMIDYLYLTRANSFERASE (EC 2.7.7.24) GLUCOSE-i -PHOSPHATE THYMIDYLYLTRANSFERASE (EC 2.7.7.24) GLUCOSE-1 -PHOSPHATE THYMIDYLYLTRANSFERASE (EC 2.7.7.24) D-R(8ITOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC 2.7.7.40) OTOP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) inositol and ribitol metabolism Nucleic Acid SEQ t0 NO 287 Amino Acid SEQ ID NO 288 Identification Code Cotg NT Start NT Stop Function RXA01887 RX087 GR00539 4219 3209 MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) 2007203042 29 Jun 2007 Table I (continued) NT Start NT Stop Function Nucleic Acid Amino Acid SEQ I0 NO SEQ ID NO Identification Code Conaig RXN0001 3 F RXA00013 RXA0 1099 RXN01 332 F RXA01332 RXAO1 632 RXA0 1633 RXN01 406 RXN01 630 RXN00528 RXN03057 F RXA02902 RXA00251 WV0048 GROO002 GR00306 WV0273 GR00388 GR00454 GROO454 VV0278 WO0050 WV0079 WV0028 GRi 0040 GROO038 7966 3566 6328 579 552 2338 3380 2999 48113 23406 7017 10277 931 8838 4438 5504 4 4 33.42 4462 1977 47037 22318 7688 10948 224 MYO-INOStTOL-1(OR 4)-MONOPHOSPR-ATASE 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.A.1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC 1. 1.18) MYO-INOSITOL 2-DEHYDROGENASE (EC 1.1.1.18)' MYO-INOSITOL.1 -PHOSPHATE SYNTHASE (EC 5.5.1.4) MYO-INOSITOL 2-DEHYDROGENASE (EC 1. 18) GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) RIBITOL 2-DEHYDROGENASE (C 1.1.1.56) Utilization of sugars Nucleic Acid SEQ 10 NO 315 317 319 321 323 325 327 329 331 Amino Acid SEQ ID NO 316 318 320 322 324 326 328 330 332 Identification Code RXN02654 F RXA02654 RXNO1 049 F RXA01049 F RXA01050 RXA00202 RXN00872 F RXA00872 RXN00799 333 334 F RXA00799 RXA00032 RXA02528 RXN00316 F RXAOO3O9 RXN00310 F RXA00310 RXA00041 RXA02026 RXA02061 Contig.

WV0090 GR00752 W0079 GR00296 GR00296 GRO0032 W0127 GR00240 W0009 GR00214 GROOD03 GR00725 W0006 GRO0053 WV0006 GR00053 GROO007 GRO0615 GRO0626 NT Start NT Stop 12206 13090 7405 8289 9633 11114 1502 492 1972 1499 1216 275 6557 5604 565 1086 581477 56834 1 1584 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) GLUCONOKINASE (EC 2.7.1.12) 0-RIBOSE-BINDING PERIPLASMIC PROTEIN PRECURSOR FRUCTOKINASE (EC 2.7.1.4) FRUCTOKINASE (EC 2.7.1.4) PERIPLASMIC BETA-GLUCOSIDASEBETA-XYLOSIDASE PRECURSOR (EC 3.2.1.21) (EC 3.2.1.37) PERIPLASMIC BETA-GLUCOSIDASEIBETA-XYLOSIDASE PRECURSOR (EC 3.2.1.21) (EC 3.2.1.37) MANNITOL 2-IJEHYDROGENASE (EC 1.1. 1.67) FRUCTOSE REPRESSOR Hypothetical Oxidoreductase (EC 111- GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) GLUCOSE--FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) SUCROSE-6-PHOSPHATE HYDROLASE (EC 3.2.1.26) 343 344 345 346 12028 6880 7035 316 6616 735 1246 725 1842 10520 7854 8180 5 7050 301 5 6 349 2007203042 29 Jun 2007 Table 1 (continued) Nucleic Acid Amino Acid Identification Code Contig NT Start NT Stop Function SEQ ID NO SEQ 1D NO 353 354 RXN01369 WV0124 595 1776 MANNOSE-6-PHOSP-ATE ISOMERASE (EC 5.3.1.8) 355 356 F RXAO 1369 GROO39B 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 RXA0261 1 GR00743 1 1752 1,4-ALPHA-GLUCAN BRANCHING ENZYME (EC: 2.4.1.18) 361 362 RXA02612 GR00743 1793 3985 1,4-ALPHA-GLUCAN BRANCHING ENZYME (EC 2.4.1.18) 363 364 RXN01684 VV0 184 1 1890 GLYCOGEN DEBRANCHING ENZYME (EC, 2.4.1.25) (EC 3.2.1.33) 365 366 F RXA01884 GR00539 3 1475 GLYCOGEN DEBRANCHING ENZYME (EC 2.4.1.25) (EC 3.2.1.33) 367 368 RXA01111 GR00306 16981 17427 GLYCOGEN OPERON PROTEIN GLGX (EC 369 370 RXN01550 WV0143 14749 16260 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 371 372 F RXAOI1550 GROD431 3 13.46 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 373 374 RXN021DO WV0318 2 2326 GLYCOGEN 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 GLYCOGEN PHOSPHORYLASE (EC 2.4.1.1) 379 360 RXA02147 GR00639 15516 16532 ALPHA-AMYLASE (EC 3.2.1.1) 381 382 RXAO1478 GR00422 10517 12352 GLUCOAMYLASE GI AND G2 PRECURSOR (EC; 3.2.1.3) 383 38.4 RXA01888 GR00539 4366 4923 GLUCOSE-RESISTANCE AMYLASE REGULATOR 385 386 RXN01927 VVO 127 50623 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 GR00762 747 4 RIBOKINASE (EC 2.7.1.15) 391 392 RXA02797 GR00778 1739 2641 RIBOKINASE (EC 2.7.1.15) 393 394 RXA02730 GR00762 1768 731 RIBOSE OPERON REPRESSOR 395 396 RYA02551 GR00729 2193 2552 6-PHOSPHO-BETA-GLUCOSIDASE (EC, 3.2.1.86) 397 398 RXA01325 GR00385 5676 5005 DEOXYRIBOSE-PHOSPHATE ALDOLASE (EC 4.1.2.4) 399 400 RXA00195 GROO030 543 1103 1 -deoxy-D-xylulose 5-phosphate reductolsamnerase (EC 1.1. 401 402 RYA00196 GR00030 1094 1708 1 -deoxy-D-xylulose 5-phosphata reductoisomerase (EC 1. 1. 403 404 RXN01 562 WV0191 1230 3137 1-DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE 405 406 F RXA0 1562 GR00436 2 1039 1 -DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE 407 408 F RX(AOI1705 GR00480 971 1573 1.-DEOXYXYLULOSE-5-PHOSPHATE SYNTHASE 409 410 RXN00879 WV0099 8763 6646 4-ALPHA-GLUCANOTRANSFERASE (EC 2,.4.1.25).

411 412 F RXA00879 GR00242 5927 3828 4.ALPHA-GLUCANOTRANSFERASE (EC 2.4.1.25), amylomahase 413 414 RXN00O43 W01l19 3244 2081 N-ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE (EC 3.5.1.25) 415 416 F RXA00043 GROO007 3244 2081 N-ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE (EC 3.5.1.25) 417 418 RXN01752 WV0127 35265 33805 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 419 420 F RXA0 1839 GR00520 1157 51 N.ACETYLGLUCOSAMINYLTRANSFERASE (EC 421 422 RXAOI 859 GR00529 1473 547 N-ACETYLGLUCOSAMINYLTRANSFERASE (EC, 423 424 RXA00042 GROO007 2037 1279 GLUCOSAMINE-6-PHOSPHATE ISOMERASE (EC 5.3.1.10) 425 426 RXA01 482 GR00422 17271 15397 GLUCOSAMINE-FRUCTOSE.-PHOSPHATE

AMINOTRANSFERASE

(ISOMERIZING) (EC 2.6.1.16) 427 428 RXN03179 WV0336 2 667 UR0ONATE ISOMERASE (EC 5.3.1.12) 429 430 F RXA02872 GRIO013 675 4 URONATE ISOMERASE, Glucuronate isomerase (EC 5.3.1.12) 431 432 RXN03180 WV0337 672 163 URONATE ISOMERASE (EC 5.3.1.12) 433 434 F RXA02873 GR10014 672 163 URONATE ISOMERASE, Glucuronate isomerase (EC 5.3.1.12) 435 436 RXA02292 GR00662 1611 2285 GALACTOSIDE 0-ACETYLTRANSFERASE (EC 2.3.1.18) 437 438 RXA02666 GR00753 7260 64 '93 D-RIBITOL-5-PHOSPHATE CYTIDYLYLTRANSFERASE (EC 2.7.7.40) 439 440 RXA00202 GR00032 1216 275 D.RISOSE-BINDING PERIPLASMIC. PROTEIN PRECURSOR 441 442 RXA02440 GR00709 5097 4258 0-RIBOSE-BINDING PERiPLASMIC PROTEIN PRECURSOR 2007203042 29 Jun 2007 Table 1 (continued) Nucleic Acid Amino Acid SEQ ID NO SE ID NO 443 444 445 4.46 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 Identification Code RXN01 569 F RXA01 569 F RXA02055 RXA00825 RYA02054 RXN00427 F RXA00427 RXA00327 RXA00328 RXA00329 RXN01 554 RXN03015 RXN03056 RXN03030 RXN00401 RXN02125 RXN00200 RXN0 1175 RXN01 376 RXN01 631 RXN01 593 RXN00337 RXS00584 RXS02574 RXS03215 F RXA01915 RXS03224 F RXA60038 RXC00233 RXCD0236 RXCOO27i RXC00338 RXC00362 RXCO0412 RXC00526 RXCO1 004 RXCO1017 RXCO1 021 RXCO 1212 RXCO1 306 RXCOI 366 RXCO1372 Contig. NT Star NT Stop Function WV0009 41086 42444 dTDP-4-DEHYDRORHAMNOSE REDUCTASE (EC 1.1.1.133) GR00438 2 427 OTDP-4-OEHYORORHAMNOSE REOUCTASE (EC 1.1.1.133) GR00624 7122 8042 DTDP-4-DEHYORORHAMNOSE REOUCTASE (EC 1.1.1.133) GR00222 222 1154 DTOP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) GR00624 6103 7119 OTOP-GLUCOSE 4,6-DEHYDRATASE (EC 4.2.1.46) WVO 112 7004 6219 dTDP-RHAMNOSYL TRANSFERASE RFBF (EC GR00098 1591 2022 DTDP-RHAMNOSYL TRANSFERASE RFBF (EC GR00057 10263 9880 PROTEIN ARAJ GR007 11147 10656 PROTEIN ARAJ GR00057 12390 11167 PROTEIN ARAJ WV01 35 28686 .26545 GLUCAN ENDO- 1.3-BETA-GLUCOSIOASE Al PRECURSOR (EC 3.2.1.39) WV0063 289 8 UDP-GLUCO iSE 6-DEHYDROGENASE (EC 1.1.1.22) WV0028 6258 6935 PUTATIVE HEXULOSE-6-PHOSPHATE ISOMERASE (EC WV0009 57006 56443 PERIPLASMIC BETA-GLUCOSIDASEJBETA-XYLOSIOASE PRECURSOR (EO 3.2.1.21) (EC 3.2.1.37) WV0025 12427 11489 5-DEHYDR0-4-DEOXYGLUCARATE DEI-YORATASE (EC 4.2.1.41) WV0102 23242 22442 ALDOSE REDUCTASE (EC 1.1.1.21) VV01 81 1679 5116 arabinosyt transferase subunit B (EC W0017 39688 38303 PHOSPHO-2-DEHYORO-3-DEOXYHEPTONATE ALOOLASE (EC 4.1.2.15) W00911 5610 4750 PUTATIVE GLYCOSYL TRANSFERASE WBIF WO0050 47021 46143 PUTATIVE HEXULOSE-6-PHOSPHATE ISOMERASE (EC WV0229 13274 12408 NAGD PROTEIN VV0197 20369 21418 GALACTOKINASE (EC 2.7.1.6) WV0323 5516 6640 PHOSPHO-2-DEIIYDRO-3-DEOXYHEPTONATE ALDOLASE (EC 4.1.2.15) BETA-HEXOSAMINIDASE A PRECURSOR (EC 3.2.1.52) GLUCOSE-FRUCTOSE OXIDOREDUCTASE PRECURSOR (EC 1.1.99.28) GR00549 1 1008 GLUC OSE-FRUCTOSE OXIDOREOUCTASE PRECURSOR (EC 1.1.99.28) CYCLOMALTODEXTRINASE (EC 3.2.1.54) GROOO06 1417 260 CYCLOMALTODEXTRINASE (EC 3.2.1.54) protein involved in sugar metabolism Membrane Lipoprotein Involved in sugar metabotism Exported Protein involved in ribose metabolism protein Invotved in sugar metabolism Membrane Spanning Protein Involved In metabolism of diols Amino Acid ABC Transporter ATP-Blnding Protein involved in sugar metabolism ABC Transporter ATP-Binding Protein Involved In sugar metabolism Membrane Spanning Protein involved in sugar metabolism Cytosollc Protein involved In sugar metabolism Cylosolic Kinase Involved in metabolism of sugars and thiamin ABC Transporter ATP-Binding Protein involved in sugar metabolism Membrane Spanning Protein involved in sugar metabolism Cylosolic Protein Involved in sugar metabolism Cytosolic Protein involved In sugar metabolism 2007203042 29 Jun 2007 Table i (continued) NT Start NT Sto- Function Nucleic Acid SEQ ID NO 527 529 531 533 535 537 539 541 Amino Acid SEQ ID NO 528 530 532 534 536 538 .540 542 Identification Code Cot.

RXCO1 659 RXCO1663 RXCO 1693 RXCO 1703 RXC02 254 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 TCA-cycle Nucleic Acid Amino Acid Identification Code SEQ ID NO SEQ ID NO 545 546 RXA02175 547 548 RXA02621 549 550 RXN00519 551 552 F RXA00521 553 554 RXN02209 555 556 F RXA02209 557 558 RXN02213 559 560 F RXA02213 561 562 RXA02056 563 564 RXA01745 565 566 RXA00782 567 568 RXA00783 569 570 RXN01695 571 572 F RXAO 1615 573 574 F RXA01695 575 576 RXA00290 577 578 RXN01048 579 580 F RXA01048 581 582 F RXA00290 583 584 RXN03101 585 586 RXN02046 587 588 RXN00389 GR00641 GR07416 W01 44 GR00133 W0304 GR00648 W0305 GR00649 GR00625 GR00495 GR00206 GR00206 W0139 GR00449 GR00474 GR00046 W0079 GR00296 GR00046 W0066 W0025 W0025 NT Start NT Stop Function 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 DEHYDROCENASE (NADP) (EC 1. 1. 1.42) ISOCITRATE DEHYDROGENASE [NADPJ (EC 1. 1. 1.42) ACONITATE HYDRATASE (EC 4.2.1.3) ACONITATE HYDRATASE (EC 4.2.1.3) ACO0ITATE I-YDRATASE (EC 4.2.1.3) ACONITATE HYDRATASE (EC 4.2.1.3) 2-OXOGLUTARATE DEHYDROGENASE El COMPONENT (EC 1.2.4.2) DIHYDROLIPOAMIDE SUCCINYLTRANSFERASE COMPONENT (E2) OF 2-OXOCLUTARATE DEHYDROGENASE COMPLEX (EC 2.3.1.6 1) SUCCINYL-COA 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 DEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) L-MALATE DEHYDROGENASE (ACCEPTOR) (EC 1.1.99.16) MALIC ENZYME (EC 1. 1. 1.39) MALIC ENZYME (EC 1.1.1.39) MALIC ENZYME (EC 1.1.1.39) MALIC ENZYME (EC 1. 1. 1.39) D01-YDROLIPOAMIDE 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 2007203042 29 Jun 2007 Glyoxylate bypass Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 589 591 593 595 597 599 Amino Acid Identification Code SEQ ID NO 590 RXN02399 592 F RXA02399 594 RXN02404 596 F RXA02404 598 RXA01089 600 RXA01886 C;onte W0176 GR00699 WV0176 GR00700 CR00304 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) GLYOXYLATE-INDUCED PROTEIN GLYOXYLATE-INDUCED PROTEIN Methyicitrate-pathway Nucleic Acid Amino Acid Identification Code SEQ ID NO SEQ IDNO 600 602 RXN03117 601 604 F RXA00406 603 606 F RXA00514 605 608 RXA00512 607 610 RXA00518 609 612 RXA01077 611 614 RXN03144 613 616 IFRXA02322 615 618 RXA02329 617 620 RXA02332 619 622 RXN02333 621 624 IF RXA02333 623 626 RXA00030 Methyl-Malonyl-CoA-Mutases Contig W0092 GR00090 GR00 130 GR001 30 GR00131 GR00300 W01 41 GR00668 GR00669 GR00671 WV0141 GR00671 GR00003 NT Start NT Stop Function 1576 4 1576 4 2773 6017 901 5 5 764 1815 1902 9979 2-methylisoctrate 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 (E0 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylisocitrate synthase (EC 2-methylcitrate synthase (EC 4.1.3.31) methylisacitrate lyase (EC 4.1.3.30) methylisocitrate lyaso (E0 4.1.3.30) LACTOYLGLUTATHIONE LYASE (EC 4.4.1.5) Nucleic Acid SEQ ID NO 625 627 629 Amino Acid Identification Code SEQ ID NO 628 RXN00148 630 F RXA00148 632 RXA00149 W0O167 CR00023 CR00023 NT Start NT Stop Function 12059 5 2009 METtIYLMALONYL-OA MUTASE ALPHA-SUBUNIT (EC 5.4.99.2) METHYLMALONYL-OA MUTASE ALPHA-SUBUNIT (EC 5.4.99.2) METHYLMALONYL-OA MUTASE BETA-SUBUNIT (EC 5.4.99.2) 2007203042 29 Jun 2007 Table 1 (continued) Others Nucleic Acid Amino Acid Identification Code Conlig NT Start NT Stop Function SEQ ID NO SEQ ID NO 631 634 RXN00317 W0197 26879 27532 PHOSP-OGLYCOLATE PHOSP-ATASE (EC 3.1.3.18) 635 636 F RXA00317 GRO0055 3.44 6 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) 637 638 RXA02196 GR00645 3956 3264 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) 639 640 RXN02461 WV0124 14236 14643 PHOSPHOGLYCOLATE PHOSPHATASE (EC 3.1.3.18) Redox Chain Nucleic Acid Amino Acid Identification Code Cni. NT Start NT Stop Function SEQ ID NO SE ID NO 641 642 RXN01744 W0174 2350 812 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 643 644 F RXA00055 GROO008 11753 11890 CYTOCHROME 0 UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 645 646 F RXA0 1744 GR00494 2113 812 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT I (EC 1.10.3.-) 647 648 RXA00379 GRO0082 212 6 CYTOCHROME C-TYPE BIOGENESIS PROTEIN OCQA 649 650 RXA00385 GR00083 773 435 CYTOCHROME C-TYPE BIOGENESIS PROTEIN CCDA 651 652 RXA01 743 GR00494 806 6 CYTOCHROME D UBIQUINOL OXIDASE SUBUNIT It (EC 1.10.3.-) 653 654 RXN02480 W0084 31222 29567 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 655 656 F RXA01919 GROO550 288 4 CYTOCHROME C OXIDASE SUBUNIT I (EC 1.9.3.1) 657 658 F RXA02480 GR00717 1449 601 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 659 660 F RXA02481 GR00717 1945 1334 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 661 662 RXAO2140 GR00639 7339 8415 CYTOCHROME C OXIDASE POLYPEPTIDE 11 (EC 1.9.3.1) 663 664 RXA02142 GR00639 9413 10063 CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9.3.1) 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 GR00032 24965 24015 ELECTRON TRANSFER FLAVOPROTEIN ALPHA-SUBUNIT 681 682 RXA00225 GR00032 25783 24998 ELECTRON TRANSFER FLAVOPROTEIN BETA-SUBUNIT 683 684 RXN00606 WV0192 11299 9026 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 685 686 F RXA0O606 GROO160 121 1869 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 687 688 RXN00595 W0192 8642 7113 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 689 690 F RXA00608 GROO160 2253 3017 NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) 691 692 RXA00913 GR00249 3 2120 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 693 694 RXA00909 GR00247 2552 3406 NADH DEHYDROGENASE I CHAIN L (EC 1.6.5.3) 695 696 RXA00700 GROO 182 846 43 NADH-UBIOUINONE OXIDOREDUCTASE CHAIN 2 697 698 RXN00483 W0086 44824 46287 NADH-UBIQUINONE OXIDOREDUCTASE 39 KO SUBUNIT PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) 2007203042 29 Jun 2007 Table 1 (continued) NT Start NT Stop Function Nucleic Acid SEQ ID NO 699 Amino Acid SEQ ID NO 700 Identification Code F RXA00483 RXA01534 RXA0028 RXA02741 RXN02560 F RXA02560 RXA01311 RXN03014 F RXAO091 0 RXN01895 F RXA01895 RXA00703 RXN00705 F RXA00705 RXN00388 F RXA00388 F RXA00386 RXA00945 RXN02556 F RXA02556 RXA01 392 RXAOO800 RXAO2143 RXN03096 RXN02036 RXN02765 RXN02206 RXN02554 GRO01 19 GROD427 GRD0046 GR00763 wolal1 GR00731 GR00380 W0058 GR00248 W01 17 GROD543 GROO1 83 W0005 GR00184 W0025 GR00085 GROO08.4 GR00259 V0 101 GR00731 GRO0408 GR00214 GR00639 WV0058 W01 76 W03 17 W0302 W0 101 19106 20569 1035 26.46 9585 9922 6339 1611 1273 3 955 2 2556 61111 1291 2081 969 514 1876 5602 2019 2297 2031 10138 405 32683 3552 1784 4633 547 1636 8620 10788 7160 865 368 1259 5 817 271 5197 407 3091 667 5 2847 6759 3176 3373 3134 11025 4 33063 2794 8.49 4010 NAOH-UBIQUINONE OXIDOREOUCTASE 39 KD SUBUNIT PRECURSOR (EC 1.6.5.3) (EC 1.6.99.3) NADH-DEPENDENT FMVN OXYDOREDUCTASE QUINONE OXIDOREDUCTASE (EC 1.6.5.5) QUINONE OXIDOREDUCTASE (EC 1.6.5.5) NADPH-FLAVIN OXIDOREDUCTASE (EC 1.6.99.-) NAOPH-FLAVIN OXIDOREDUCTASE (EC 1.6.99.-) SUCCINATE DEHYOROGENASE IRON-SULFUR PROTEIN (EC 1.3.99.1) NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) Hydrogenase subunits NADH DEHYOROGENASE (EC 1.6.99.3)

DEH-YDROGENASE

FORM ATE DEHYDROGE.NASE ALPHA CHAIN (EC 1.2.1.2) FDHb PROTEIN FDHD PROTEIN CYTOCHROME C BIOGENESIS PROTEIN CCSA essential protein similar to cytoclirome c RESO PROTEIN, essential protein similar to cytochrome c biogenesis protein putative cytochrome oxidase FLAVOI-EMOPROTEIN I DIHYOROPTERIDINE REDUCTASE (EC 1.6.99.7)

FLAVOHEMOPROTEIN

GLUTATHIONE S-TRANSFERASE (EC 2.5.1.18) GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE (EC 11.2.11.1) QCRC PROTEIN, menaquinot:cytochrome c oxidoreductase NADH DEHYDROGENASE I CHAIN M (EC 1.6.5.3) NAOH-UBIOUINONE OXIDOREDUCTASE CHAIN 4 (EC 1.6.5.3) Hypothetical Oxidlorductase Hypothetical Oxidoreductase Hypothetical Oxidoreductase (EC 111- 733 734 735 736 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 RXA01204 RX)AO1 201 RXN01 193 F RXAO 1193 F RXA01203 W01 21 GR00345 GR00344 W0175 GR00343 GROO344 NT Start NT Stop 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 SYNTH-ASE 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) 2007203042 29 Jun 2007 Nucleic Acid SEQ ID NO 767 769 771 773 775 777 Table I (continued) NT Start NT Stop Function Amino Acid Identification Code Cog.

SEQ I0 NO 768 RXN02821 WV0121 770 F RXA02821 GRO08I 772 RXA01200 GROO3 774 RXA01 194 GRO03 776 RXA01 202 GR0034 778 RXN0l244 1AJfno 324 02 139 14 2 13 770 14 2375 I 4923 ATP SYNTHASE C CHAIN (C 3.6.1.34) ATP SYNTI-ASE 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 Cor.

SEQ ID NO SEQ ID NO 779 780 R.XN00684 WV0005 781 782 RXN00387 VV0025 NT Start NT Stop Function 29864 28581 1150 2004 CYTOCHROME P450 116 (EC Hypothetical Cylochrome c Biogenesis Protein 2007203042 29 Jun 2007 TABLE 2 Excluded Genes GenBanklu Gene Name Gene Function Reference Accession No.

A09073 ppg PhosphoenolI pyruvate carboxylase Bachmann, B. et al "DNA fragment coding for phosphoenolpyruvat corboxylase, recombinant DNA carrying said fragment, strains carrying the recombinant DNA and method for producing L-aminino acids using said strains," Patent: EP 0358940-A 3 03/21/90 A45579, Threonine dehydratase Moeckel, B. et "Production of L-isoleucine by means of recombinant A45581, micro-organisms with deregulated threonine dehydratase," Patent: WO A45583, 9519442-A 5 07/20/95 A45585 A45587 AB0O3 132 murC; ftsQ; ftsZ Kobayashi, M. et al. "Cloning, sequencing, and characterization of the ftsZ gene from coryneform bacteria," Biochem. Biophys. Res. Comm un., 236(2):383-388 (1997) ABO 15023 murG; fisQ Wachi, M. et al. "A murC gene from Corynefo-m bacteria," Appi. Microbiol BiolechnoL, 5](2):223-228 (1999) ABO 18530 dtsR Kimura, E. et al. "Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium laclofermentum," Biosci. Biolechnol Biochem., 60(10): 1565-1570 (1996) ABO 18531 dtsR]; dtsR2 AB020624 muri D-glulamale racemase AB023377 tkt transketolasc AB024708 gltB; gID Glutamine 2-oxoglutarate aminotransferase large and small subunits AB025424 acn aconitase A B0277 14 rep Replication protein AB027715 rep; aad Replication protein; aminoglycoside adenyltransferase AF005242 argC dehydrogenase AF005635 gInA Glutamine AF030405 hisF cyclase AF030520 argG Argininosuccinate synthetase AF031518 argF Ornithine carbamolytransferase AF036932 aroD 3-dehydroquinate AF038548 pyc Pyruvate 2007203042 29 Jun 2007 2 (continued) AF038651I dciAE; apt; rel Dipept ide-bin ding protein; adenine Wehmeier, L. et Ial. ITh rcle: of1 he Gorynebacteriumn giutamicum rel gene in AF041436 argR Arginine repressor A F-45998 impA Inositol monophsht hopatase AF048764 argH Argininosuccinate lyase A F049897 argC; argi; agB N-acctylglutamlpI spaF rductase; argD; argF; argR; ornithine acetyltransferase;

N-

argG; argH acetylgiutamate kinase; acetylorrithine transminase; omnithine carbamoyltransferase; arginine repressor; argininosuccinate synthase; lyase AFOSO 109- inhA Enoyl-acyl carrier protein reductase A F050166 h is ATP phosphoribosy-transferase 1846 hisA Phlosphoribosylform imino-5-amimo- Iphosphoribosyl-im idazolecarboxam ide AF052652 metA Homoserine O-acetyl transt'erase Park, S. et al. "Isolation and analysis of me'tA amethionnn bi A AF053071I aroB E Dehydroquinate synthetase AF060558 hisH G Glutamine amidotransferase encoding homoserine acetyltransferase in Corynebacterium glutam icum," McI.

Cells., 8(3):286-294 (1998) YAF086704 fist AF114233 JaroA Phosphoribosyl-ATPpyrophosphohydrolase 5-enoipyruvyishikimate 3-phosph-ate synthase I I U I f pariu L-aspartate-alpha-decarboxylse precursor Dusch, N. et al. "Expression of the Corynebacterium glutamicumn panD gene encoding L-asparlate-alpha-decarboxylase leads to pantochenate overproduction in Eschcrichia coli," App/. Environ. lviicrobiol., 65(4)1530- 1539 (1999) -I I 1&It.'*J1I0 aIoL), aror.

3-dehydroquinase; shikimate dehydrogenase AF124600 aroC; aroK; aroB; pepQ Chorismate synthase; shikimate -kinase; 3dehydroquinate synthase; putative cytoplasmic peptidase A F145897 inhA A17145898 inhA 2007203042 29 Jun 2007 Tble2 oninued) AJO0 1436 ectP Transport of ectoine, glycine betaine, Peter, H. et al. "Corynebacterium glutamnicum is equipped with four secondary proline carriers for compatible solutes: Identification, sequencing, and characterization of the proline/ectoine uptake system, Prop, and the ectoine/proline/glycine carrier, EcIP," J Bacteriol, 180(22):6005-6012 (1998) AJ004934 dapD Tetrahydrod ipicol in ate succinylase Wehrmann, A. et al. "Different modes of diaminopimelate synthesis and their (incomplete) role in cell wall integrity: A study with Corynebacterium glutamicum,"J 180(12):3 159-3165 (1998) AJ007732 ppc; secG; amt; ocd; Phosphoenolpyruvate-carboxylase; high soxA affinity ammonium uptake protein; putative ornith ine-cyclodecarboxylase; sarcosine AJOIO3 19 ftsY, glnB, ginD; srp; Involved in cell division; PH1 protein; Jakoby, M. et al. "Nitrogen regulation in Corynebacteriumn glutamicum; amtP uridylyltransferase (uridylyl-removing Isolation of genes involved in biochemical characterization of corresponding enzmye); signal recognition particle; low proteins," FEMS Microbial., 173(2):303-3)10 (1999) affinity ammonium uptake AJ 132968 cat Chloramphenicol aceteyl transferase A)224946 mqo L-malate: quinone oxidoreductase Molenaar, D. et al. "Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Corynebacterium Eur. J Biachem., 254(2):395-403 (1998) AJ238250 ndh NADH 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 mass polypeptide," Biachemisiry, 37(43):15024-15032 (1998) D17429 Transposable element 1S31831 Vertes et al."lsolation and characterization ofIS3183l1, a transposable element from Corynebacteriumn glutamicum," Mol. MicrobioL, 11 (4):739-746 (1994) D84 102 odhA 2-oxoglutarate dehydrogenase Usuda, Y. et al. "Molecular cloning of the Corynebacteriuni glutamicum (Brevibacterium lactofermentumn AJ 12036) odhA gene encoding a novel type 2-oxoglutarale dehydrogenase," Microbiology, 142:3347-3354 (1996) E01358 hdh; hk H-omoserine dehydrogenase; homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleucine," Patent:. JP 198723 2392-A 1 10/12/87 E01359 Upstream of the start codon of homoserine Katsumata, R. et al. "Production of L-thereonine and L-isoleticine," Patent: JP gene 1987232392-A 2 10/12/87 E0 1375 Tryptophan operon E0 1376 trpL; trpE Leader peptide; anthranilate synthase Matsui, K. et al. "Tryptophan operon, peptide and protein coded thereby, utilization of tryptophan operon gene expression and production of Patent: JP 1987244382-A 1 10/24/87 2007203042 29 Jun 2007 Table 2 (continued) E01377 Promoter and operator regions of Matsui, K. et at. "Tryptophan operon, peptide and protein coded thereby, tryptophan operon utilization of trptophan operon gene expression and production of tryptophan," Patent: JP 1987244382-A 1 10/24/87 E03937 Biotin-synthase Hatakeyama, K. et at. "DNA fragment containing gene capable of coding biotin synthetase and its utilization," Patent: JP 1992278088-A 1 10/02/92 E04040 Diamino pelargonic acid aminotransferase Kohama, K. et at. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization," Patent: JP 1992330284-A I 11/18/92 E04041 Desthiobiotinsynthetase Kohama, K. et at. "Gene coding diaminopelargonic acid aminotransferase and desthiobiotin syn(hetase and its utilization," Patent: JP 1992330284-A I I11/ 18/90 E04307 Flavum aspartase Kurusu, Y. et at. "Gene DNA coding aspartase and utilization thereof," Patent: 1993030977-A 1 02/09/93 E04376 Isocitric acid lyase Katsumata, R. et at. "Gene manifestation controlling DNA," Patent: JP 1993056782-A 3 03/09/93 E04377 Isocitric adid lyase N-terminal fragment Katsumata, R. et at. "Gene manifestation controlling DNA," Patent: JP 3 03/09/93 E04484 Prephenate dehydratase Sotouchi, N. et at. "Production of L-phenylalanine by fermentation," Patent: JP 1993076352-A 2 03/30/93 E05108 Aspartokinase Fugono, N. et at. "Gene DNA coding Aspartokinase and its use," Patent: JP 1993184366-A 1 07/27/93 112 Dihydro-dipichorinate synthetase Hatakeyama, K. et at. "Gene DNA coding dihydrodipicotinic acid synthetase and its use," Patent: JP 1993184371-A 1 07/27/93 E05776 Diaminopimelic acid dehydrogenase Kobayashi, M. et at. "Gene DNA coding Diaminopimelic acid dehydrogenase its use," Patent: JP 1993284970-A 1 11/02/93 E05779 Threonine synthase Kohama, K. et al. "Gene-DNA coding threonine synthase and its use," Patent: 1993284972-A 1 11/02/93 E061 10 Prephenate dehydratase Kikuchi, T. et at. "Production of L-phenylalanine by fermentation method," Patent: JP 1993344881-A 1 12/27/93 E061 1l Mutated Prephenate dehydratase Kikuchi, T. et at. "Production of L-phcnylatanine by fermentation method," Patent: JP 1993344881I-A 1 12/27/93 E06146 Acetohydroxy acid synthetase Inui, M. et at. "Gene capable of coding Acetohydroxy acid synthetase and its Patent: JP 1993344893-A 1 12/27/93 E06825 Aspartokinase Sugimoto, M. et at. "Mutant aspartokinase gene," patent: JP 1994062866-A 1 E06826 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A I 2007203042 29 Jun 2007 Table 2 (continued) E06827 Mutated aspartokinase alpha subunit Sugimoto, M. et al. "Mutant aspartokinase gene," patent: JP 1994062866-A 1 03/08/94 E07701 secY Honno, N. et al. "Gene DNA participating in integration of membraneous protein to membrane," Patent: JP 1994169780-A 1 06/21/94 E08177 Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08178, Feedback inhibition-released Aspartokinase Sato, Y. et al. "Genetic DNA capable of coding Aspartokinase released from E08179, feedback inhibition and its utilization," Patent: JP 1994261766-A 1 09/20/94 E08180, E08181, E08182 E08232 Acetohydroxy-acid isomeroreductase Inui, M. et al. "Gene DNA coding acetohydroxy acid isomeroreductase," Patent: JP 1994277067-A .1 10/04/94.

E08234 secE Asai, Y. ct al. "Gene DNA coding for translocation machinery of protein," Patent: JP 1994277073-A I 10/04/94 E08643 FT aminotransferase and desthiobiotin Hatakeyama, K. et al. "DNA fragment having promoter function in synthetase promoter region coryneform bacterium," Patent: JP 1995031476-A I 02/03/95 E08646 Biotin synthetase Hatakeyama, K. et al. "DNA fragment having promoter function in coryneform bacterium," Patent: JP 1995031476-A 1 02/03/95 E08649 Aspartase Kohama, K. et al "DNA fragment having promoter function in coryneform bacterium," Patent: JP 1995031478-A 1 02/03/95 E08900 Dihydrodipicolinate reductase Madori, M. et al. "DNA fragment containing gene coding Dihydrodipicolinate acid reductase and utilization thereof," Patent: JP 1995075578-A I 03/20/95 E08901 Diaminopimelic acid decarboxylase Madori, M. et al. "DNA fragment containing gene coding Diaminopimelic acid decarboxylase and utilization thereof," Patent: JP 1995075579-A 1 03/20/95 E12594 Serine hydroxymethyltransferase Hatakeyama, K. et al. "Production of L-trypophan," Patent: JP 1997028391-A 1 02/04/97 E12760, transposase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: E12759, JP 1997070291-A 03/18/97 E12758 E12764 Arginyl-tRNA synthetase; diaminopimelic Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: acid decarboxylase JP 1997070291-A 03/18/97 E12767 Dihydrodipicolinic acid synthetase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 E12770 aspartokinase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 El2773 Dihydrodipicolinic acid reductase Moriya, M. et al. "Amplification of gene using artificial transposon," Patent: JP 1997070291-A 03/18/97 2007203042 29 Jun 2007 Table 2 (continued) E1 3655 G lucose-6- phosphate dehydrogenase Hatakeyama, K. et al. "GlIucose-6- phosphate dehydrogenase and DNA capable coding the same," Patent: JP 1997224661I-A 1 09/02/97 L01508 INvA Threonine dehydratase Moeckel, B. et al. "Functional and structural analysis of the threonine dehydratase of Gorynebacterium glutamicum," J. Bacteriol, 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 glutam icum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene," Uicrobiol Left., 107:223-230 (1993) L09232 IlvB; 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. Bacteriol. 175(17):5595acid isomeroreductase 5603 (1993) L 18874 PtsM Phosphoenolpyruvate sugar Fouet, Ac-t al.* "Bacillus subtilis sucrose-specific enzyme 11 of the phosphotransferase phosphotransferase system: expression in Escherichia coli and homology to enzymes 11 from enteric bacteria," PNAS USA, 84(24):8773-8777 (1987); Lee, J.K. et al. "Nucleotide sequence of the gene encoding the Corynebacteriumn glutamicum mannose enzyme 11 and analyses of the deduced protein FEMS Microbiol. Let., 119(1-2):137-145 (1994) L27 123 aceB Malate synthase Lee, H-S. et al. "Molecular characterization of aceB, a gene encoding malate synthase in Corynebacterium glutamicum," J. Microbiol Biotechnol., (1994) L27126 Pyruvate kinase Jetten, M. S. et al. "Structural and functional analysis of pyruvate kinase from Corynebacterium glutam icum," Appi. Environ. Microbial., 60(7):250 1-2507 L28760 aceA Isocitrate L35906 dtxr Diphtheria toxin repressor Oguiza, J.A. et al. 'Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR from Brevibacterium J. Bacteriol., 1 77(2):465-467 (1995) M 13774 Prephenate dehydratase Follettie, M.T. et al. "Molecular cloning and nucleotide sequence of the Corynebacterium glutam icum pheA gene," Bacterial., 167:695-702 (1986) M 16175 5SrRNA Park, et al. "Phylogenetic analysis of the coryneform bacteria by 56 rRNA sequences," J Bacterial, 169:180.1-1806 (1987) M 16663 trpE Anthranilate synthase, 5' end Sano, K. et al. "Structure and function of the trp operon control regions of Brevibacterium lactofermentumn, a glutamic-acid-producing bacterium," Gene, 52:19 1-200 (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 glutamic-acid-producing bacterium," Gene, 52:19 1-200 (1987) 2007203042 29 Jun 2007 Table 2 (continued) M25819 Phosphoenolpyruvate carboxylase O'Regan, M. et al. "Cloning and nucleotide sequence of the Phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032," Gene, 77(2):237-251 (1989) M85106 23S rRNA gene insertion sequence Roller, C. et al. "Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes," J. Gen.

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

Microbiol., 138:1167-1175 (1992) M89931 aecD; bmQ; yhbw Beta C-S lyase; branched-chain amino acid Rossol, 1. et al. "The Corynebacterium glutamicum aecD gene encodes a C-S uptake carrier; hypothetical protein yhbw lyase with alpha, beta-elimination activity that degrades aminoethylcysteine," J. Bacteriol., 174(9):2968-2977 (1992); Tauch, A. et al. "Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product," Arch. Microbiol, 169(4):303-312 (1998) S59299 trp Leader gene (promoter) Herry, D.M. et al. "Cloning of the trp gene cluster from a tryptophanhyperproducing strain ofCorynebacterium glutamicum: identification of a mutation in the trp leader sequence," Appl. Environ. Microbiol., 59(3):791-799 (1993) U 11545 trpD Anthranilate phosphoribosyltransferase O'Gara, J.P. and Dunican, L.K. (1994) Complete nucleotide sequence of the Corynebacterium glutamicum ATCC 21850 tpD gene." Thesis, Microbiology Department, University College Galway, Ireland.

U 13922 cglIM; cglIR; clglIR Putative type II 5-cytosoine Schafer, A. et al. "Cloning and characterization of a DNA region encoding a methyltransferase; putative type II stress-sensitive restriction system from Corynebacterium glutamicum ATCC restriction endonuclease; putative type I or 13032 and analysis of its role in intergeneric conjugation with Escherichia type III restriction endonuclease coli,"J. Bacteriol., 176(23):7309-7319 (1994); Schafer, A. et al. "The Corynebacterium glutamicum cgllM gene encoding a 5-cytosine in an McrBCdeficient Escherichia coli strain," Gene, 203(2):95-101 (1997) U 14965 recA U31224 ppx Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J Bacteriol., 178(15):4412-4419 (1996) U31225 proC L-proline: NADP+ 5-oxidoreductase Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., 178(15):4412-4419 (1996) U31230 obg; proB; unkdh ?;gamma glutamyl kinase;similar to D- Ankri, S. et al. "Mutations in the Corynebacterium glutamicumproline isomer specific 2-hydroxyacid biosynthetic pathway: A natural bypass of the proA step," J. Bacteriol., dehydrogenases 178(15):4412-4419 (1996) 00 2007203042 29 Jun 2007 Table 2 (continued) U31281 bioB Biotin synthase Serebriiskii, "Two new members of the blo B superfamily: Cloning, sequencing and expression of bio B genes of Methylobacillus flagellatum and ________________Corynebacteriumn glutamicum," Gene, 175:15-22 (1996) U35023 thtR; accBC Thiosulfate sulfurtransferase; acyl CoA Jager, W. et al. "A Corynebacteriumn glutamicumn gene encoding a two-domain carboxylase protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins,' Microbiol., 166(2);76-82 (1996) U43535 cmr Multidrug resistance protein Jager, W. et al. "A Corynebacterium glutamicum gene conferring multidrug resistance in the heterologous host Escherichia coli," J Bacteriol., 179(7):2449-2451I (1997) U43536 clpB Heat shock ATP-binding protein U53587 aphA-3 3'5' -am inoglycoside phos photransferase U89648 Corynebacteriumn glutamicum unidentified sequence involved in histidline biosynthesis, X04960 trpA; trpB; trpC; trpD; Tryptophan operon Matsui, K. et al. "Complete nucleotide and deduced amino acid sequences of trpE; trpG; trpL the Brevibacterium lactofermentumn tryptophan operon," Nucleic Acids Res., 13-101 14 (1986) X07563 lys A DAP decarboxylase (meso-diaminopimelate Yeh, P. et al. "Nucleic sequence of the lysA gene of Corynebacterium decarboxylase, EC 4.1.1.20) glutamicumn and possible mechanisms for modulation of its expression," Mo.

Gen. Gene., 212(I):112-1 19 (1988) X 14234 EC 4.1.1.31 Phosphoenolpyruvate carboxylase Eikmanns, BiJ. et al. "The Phosphoenolpyruvate carboxylase gene of Corynebacteriumn glutamicum: Molecular cloning, nucleotide sequence, and expression," Mo6!. Gen. Genet., 218(2):330-339 (1989); Lepiniec, L. et al.

"Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function and molecular evolution," Plant. Mo!. Bial., 21 (3):487-502 (1993) X 17313 fda Fructose- bisphosph ate aldolase Von der Osten, C.H. et al. "Molecular cloning, nucleotide sequence and finestructural analysis of the Corynebacterium glutamicumn fda gene: structural comparison of C. glutamicumn fructose-I1, 6-biphosphate aldolase to class l and class 11 aldolases," Ma!. Microbial., X53993 dapA L-2, 3-dihydrodipicolinate synthetase (EC Bonnassie, S. et al. "Nucleic sequence of the dapA gene from 4.2.1.52) Corynebacterium glutamicum," Nucleic Acids Res., 18(21):6421 (1990) X54223 AttB-related site Cianciotto, N. et al. "DNA sequence homology between att B3-related sites of Corynebacteriumn diphtheriae, Corynebacterium u Icerans, Corynebacteriumn glutamicum and the attP site of lambdacorynephage," FEMS. Micro biol, etf., 66:299-302 (1990) X54740 argS; lysA Arginyl-tRNA synthetase; Diaminopimelate Marcel, T. et al. "Nucleotide sequence and organization of the upstream region dlecarboxylase of the Corynebacterium glutamicumn lysA gene," Ma!. Microbial., 4(11): 1819- 1830 (1990) 2007203042 29 Jun 2007 2 (continued) X55994 trpL; trpE Putative leader peptide; anthranilate I-eery, D.M. et al. "Nucleotide sequence of the Corynebacterium glutamicum component I trpE gene," Nucleic Acids Res., 18(23):7 138 (1990) X56037 thrC Threonine synthase Han, K.S. et al. "The molecular structure of the Corynebacterium glutamicumn threonine synthase gene," Mot. Microbiol., 4(10): 1693-1702 (1990) X56075 attB-related site Attachment site Cianciorto, N. et al. "DNA sequence homology between att B3-related sites of Corynebacterium diphtheriae, Corynebacteriumn ulcerans, Corynebacterium glutamicum and the attP site of lambdacorynephage," FEMS. Microbiol, 66:299-302 (1990) X57226 lysC-alpha; lysC-beta; Aspartokinase-alpha subunit; Kalinowski, J. et al. "Genetic and biochemical analysis of the Aspartokinase asd Aspartokinase-beta subunit; aspartate beta from Corynebacterium glutamicum," Mo. Microbiol., 1197-1204 (1991); semnialdehyde dehydrogenase Kalinowski, J. et al. "Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum," Mo!. Gen. Genet., 224(3):317-324 (1990) X59403 gap-,pgk; tpi Glyceraldehyde-3-phosphate; Eikmanns, B.J. "Identification, sequence analysis, and expression of a phosphoglycerate kinase; triosephosphate Corynebacterium glutamnicum gene cluster encoding the three glycolytic isomerase enzymes glyceraldehyde-3 -phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomeras," J. Bacteriol.. 174(19):6076-6086 X59404 gdh Glutamnate dehydrogenase Bormann, E.R. et al. "Molecular analysis of the Corynebacterium glutamnicumn gdh gene encoding glutamate dehydrogenase," Mo!. MicrobioL., 6(3):317-326 (1992) X60312 lysI L-lysine permease Seep-Feldhaus, A.H. et al. "Molecular analysis of the Corynebacterium glutamicum lysi gene involved in lysine uptake," Mo!. UicrobioL, 5(12):2995- 3005 (1991) X66078 cop I PsI protein Joliff, G. et al. "Cloning and nucleotide sequence of the csplI gene encoding PSI1, one of the two major secreted proteins of Corynebacterium glutamicum: The deduced N-terminal region of PSI is similar to the Mycobacterium antigen complex," Mo!. Microbiol, 6(16):2349-2362 (1992) X66 112 git Citrate synthase Eikmanns, B.J. et al. "Cloning sequence, expression and transcriptional analysis of the Corynebacterium glutamicumn gItA gene encoding citrate Microbiol., 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 glutam icum," AM6. Mlicrobiol, 9(1):97-109 (1993) X69104 153 related insertion element Bonamy, C. et al. "Identification of IS 1206, a Corynebacterium glutamicumn 1S3-related insertion sequence and phylogenetic analysis," Mol. MicrobioL., 14(3):571-581_(1994) 2007203042 29 Jun 2007 2 (continued) X70959 leuA Isopropylmalate synthase Patek, M. et al. "Leucine synthesis in Corynebacteriumn glutamicum: enzyme activities, structure of leuA, and effect of leuA inactivation on lysine synthesis," App!. Environ. Microbial, 60(1): 133-140 (1994) X7 1489 lcd Isocitrate dehydrogenase (NADP+) Eikmanns, B.J. et al. "Cloning sequence analysis, expression, and inactivation of the Corynebacterium glutamicumn icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme," J Bacterial, (1995) X72855 GDHA Glutamate dehydrogenase (NADP-) X75083, mtrA 5-methyltryptophan resistance Heery, D.M. et al. "A sequence from a tryptophan-hyperproducing strain of X70584 Corynebacteriumn glutamicum encoding resistance to S-methyln-yptophan," Biaphys. Res. Comnmun., 201(3):1255-1262 (1994) X75085 recA Fitzpatrick, R. et al. "Construction and characterization of recA mutant strains of Gorynebacteriumn glutamicumn and Brevibacterium lactofermentum," AppI.

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

Bacterial., 176(12):3474-3483 (1994) X76875 ATPase beta-subunit Ludwig, W. et at. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit Anionie Van Leeuwenhoek 64:285-305 (1993) X77034 tuf Elongation factor Tu Ludwig, W. et al. "Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes," Anianie Van Leeuwenhaek 64:285-305 (1993) X77384 recA Billman-Jacobe, H. "Nucleotide sequence of a recA gene from Corynebacterium glutamicum," DNA Seq.. 4(6):403-404 (1994) X78491 aceB Malate synthase Reinscheid, DIJ et al. "Malate synthase from Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase: sequence analysis," Microbiology, 140:3099-3108 (1994) X80629 16S rDNA 16S ribosomal RNA Rainey, F.A. et al. "Phylogenetic analysis of the genera Rhodococcus and Norcardia and evidence for the evolutionary origin of the genus Norcardia from within the radiation of Rhodococcus species," Microbial, 141:523-528 (1995) X81 191 gluA; gluB; gluC; Glutamate uptake system Kronemeyer, W. et al. "Structure of the gluABCD cluster encoding the glu D glutamate uptake system of Corynebacteriumn glutamicum," J. Bacterial, 177(5):1 152-1158 (1995) X81379 dapE Succinyldiaminopimelate desuccinylase Wehrmann, A. et al. "Analysis of different DNA fragments of Corynebacteriumn glutam icumn complementing dapE of Escherichia coli, 40:3349-56 (1994) 2007203042 29 Jun 2007 Table 2 (continued) X82061 16S rDNA 16S ribosomal RNA Ruimy, R. et al. "Phylogeny of the genus Corynebacterium deduced from analyses of small-subunit ribosomal DNA sequences," Int. J. Syst. Bacteriol., 45(4):740-746 (1995) X82928 asd; lysC Aspartate-semialdehyde dehydrogenase; Serebrijski, I. et al. "Multicopy suppression by asd gene and osmotic stressdependent complementation by heterologous proA in proA mutants," J.

Bacteriol., 177(24):7255-7260 (1995) X82929 proA Gamma-glutamyl phosphate reductase Serebrijski, I. et al. "Multicopy suppression by asd gene and osmotic stressdependent complementation by heterologous proA in proA mutants," J.

Bacteriol., 177(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," Int. J. Syst. Bacteriol.. 45(4):724-728 (1995) X85965 aroP; dapE Aromatic amino acid permease; Wehrmann et al. "Functional analysis of sequences adjacent to dapE of C.

glutamicum proline reveals the presence of aroP, which encodes the aromatic amino acid transporter," J. Bacteriol., 177(20):5991-5993 (1995) X86157 argB; argC; argD; Acetylglutamate kinase; N-acetyl-gamma- Sakanyan, V. et al. "Genes and enzymes of the acetyl cycle of arginine argF; argJ glutamyl-phosphate reductase; biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early acetylornithine aminotransferase; ornithine steps of the arginine pathway," Microbiology, 142:99-108 (1996) carbamoyltransferase; glutamate Nacetyltransferase X89084 pta; ackA Phosphate acetyltransferase; acetate kinase Reinscheid, D.J. et al. "Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding 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 FI Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90357 Promoter fragment F2 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X90358 Promoter fragment F10 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) 2007203042 29 Jun 2007 Table 2 (continued) X90360 Promoter fragment P22 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology 142-1297-1309 (1996) X90361 Promoter fragment F34 Patek, M. et al. "Promoters from Corynebacterium glucamicum: cloning, molecular analysis and search for a consensus motif," Microbiology, 142:1297-1309 (1996) X 9-0362 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 Coiynebacterium 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, (1996) X90365 Promoter fragment F75 Patek, M. et al. "Promoters from Corynebacterium glutaniicum: cloning, molecular analysis and search for a consensus motif," Microbiology, (1996) X90366 Promoter fragment PFI10I 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 PP 104 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology.

(1996) X90368 Promoter fragment PP 109 Patek, M. et al. "Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif," Microbiology (1996) X935 13 amt Ammonium transport system Siewe, R.M. et al. "Functional and genetic characterization of the (methyl) ammonium uptake carrier of Corynebacterium glutamicum," J. Biol. Chemn., 0):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 solute glycine betaine," J lacteriol., 178(1 7):5229-5234 (1996) X95649 orf4 Patek, M. et al. "Identification and transcriptional analysis of the dapB-ORF2dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes in L-lysine synthesis," Biotechnol. Leif., 19:1113-1117 (1997) X96471 lysE; IysG 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," Mot.

Microbiol., 22(5):8 15-826 (1996) 2007203042 29 Jun 2007 Table 2 (continued) X96580 panB; panC; xylB 3-methyl-2-oxobutanoate Sahm, H. et al. "D-pantothenate synthesis in Corynebacterium glutamicum and hydroxymethyltransferase; pantoate-beta- use of panBC and genes encoding L-valine synthesis for D-pantothenate atanine ligase; xylulokinase overproduction," App!. Environ. Microbial., 65(5):1973-1979 (1999) X96962 Insertion sequence IS 1207 and transposase X99289 Elongation ractor P Ramos, A. et at. "Cloning, sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofermentum glutamicumn ATCC 13869)," Gene, 198:217-222 (1997) Y00 140 thrB Homoserine kinase Mateos, L.M. et at. "Nucleotide sequence of the homoserine kinase (thrB) gene of the Brevibacterium lactofermentum," Nucleic Acids Res., 15(9):3922 (1987) Y00151 ddh Meso-diaminopimelate D-dehydrogenase Ishino, S. et at. "Nucleotide sequence of the meso-diaminopimelate D- (EC 1.4.1.16) dlehydrogenase gene from Corynebacterium glutamicum," Nucleic Acids Res., (1987) Y00476 thrA Homoserine dehydrogenase Mateos, L.M. et al. "Nucleotide sequence of the homoserine dehydrogenase (thrA) gene of the Brevibacteriumn 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 glutamicum hom-thrB operon," Mo. Mlicrobial., 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 Brevibacterium Iactoferrnentum," Mo! Gen.

cell division protein Genet., 259(1):97-104 (1998) Y09 163 putP High affinity praline transport system Peter, H. et at. "Isolation of the putP gene of Corynebacterium glutamicumproline and characterization of a tow-affinity uptake system for solutes," Archi. Microbiol., 168(2):143-151 (1997) Y09548 pyc Pyruvatc carboxylase Peters-Wendisch, P.G. et at. "Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene," 144:915-927 (1998) Y09578 leuB 3-isopropylmalate dehydrogenase Patek, M. et at. "Analysis of the leuB gene from Corynebacterium App!. Microbial. Biotechnol., 50(1):42-47 (1998) Y 12472 Attachment site bacteriophage Phi- 16 Moreau, S. et at. "Site-specific integration of corynephage Phi- 16: The construction of an integration vector," Microbial., 145:539-548 (1999) Y 12537 proP Protinelectoine 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 Ibetaine carrier, EctP," J. Bacterial., 180(22):6005-6012 (1998) 2007203042 29 Jun 2007 Table 2 (continued Y13221 gInA Glutamine synthetase I Jakoby, M. et al. "Isolation of Corynebacterium glutamicum gInA gene encoding glutamine synthetase FEMS Microbiol. Let., 154(1):81-88 (1997) Y16642 Ipd 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(1):150-159 (1999) Z21501 argS; lysA Arginyl-tRNA synthetase; diaminopimelate Oguiza, J.A. et al. "A gene encoding arginyl-tRNA synthetase is located in the decarboxylase (partial) upstream region of the lysA gene in Brevibacterium lactofermentum: Regulation of argS-lysA cluster expression by arginine," J.

Bacteriol., 175(22):7356-7362 (1993) Z21502 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. Bacteriol., 175(9):2743-2749 (1993) Z29563 thrC Threonine synthase Malumbres, M. et al. "Analysis and expression of the thrC gene of the encoded threonine synthase," Appl. Environ. Microbiol., 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. Bacteriol., 178(2):550- 553 (1996) Z49823 galE; dtxR Catalytic activity UDP-galactose 4- Oguiza, J.A. et al "The galE gene encoding the UDP-galactose 4-epimerase of epimerase; diphtheria toxin regulatory Brevibacterium lactofermentum is coupled transcriptionally to the dmdR protein gene," Gene, 177:103-107 (1996) Z49824 orfi; sigB SigB sigma factor Oguiza, J.A. et al "Multiple sigma factor genes in Brevibacterium lactofermentum: Characterization of sigA and sigB," J. Bacteriol., 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 lactofermentum ATCC 13869," Gene, 170(1):91-94 (1996) A sequence for this gene was published in the indicated reference. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region: 86 TABLE 3: Corynebacterium and Brevibacteriurn Strains Which May be Used in the Practice of the Invention Brevibacterium ammoniagenes 210354 I I Brevibacterium amoiges930___ Brevibacterium ammoniagenes 19350 Brevibacterium arnron iagenes 19352 Brevibacerium ammoniagenes .19353 Brevibacterium ammoniagenes 193543_______ Brevibacterium ammoniagenes 193554___ Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 193556__ Brevibacterium arnmoniagenes 21077 Brevibacterum ammoniagenes 21553 Brevibacterium ammoniagenes 2158053__ Brevibacterium- ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brcvibacterium- flavum 21129 Brevibacterium 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 Brevibaccerium flavum BI 1477 Brevibacterium flavum BI 1478 Brevibacterium fiavum 21127 Brevibacterium flavum BI 1474 Brevibaccerium healii 15527 Brevibacterium ketoglutamicum 21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevibacterium lactofermentum Brevibacterium lactofementum 74 Brevibaccerium lactoferme'ntum 77 Brevibacterium lactofe-mentum 21798 Brevibacterium lactofermentum 21799 Brevibacterium lactofen-nentum 21800 Brevibacterium lactofermentum 21801 Brevibacterium lactofermennim B] 1470 Brevibacterium lactofermentum Bl 11471 87 Genus. -~species -7 ATCC. FERNI NRtCECT. ~Ch NK C MVI Brevibacterium lactofermentumn 21086 Brevibacterium lactofermentumn 21420 Brevibacterium lactofermentumn 21086 Brevibacterium lactofermentrm 31269 Brevibacterium linens 9174 Brevibacterium linens 1939] Brevibacterium linens 8377 Brevibacterium paraffinolyticum 11160 Brevibacterium Spec. Brevibacterium Spec. _____717.73 Brevibacterium Spec. 14604 Brevibacterium Spec. 21860 Brevibacterium Spec. 21864 Brevibacterium Spec. 21865 Brevibacterium Spec. 21866 Brevibacterium Spec. 19240 Corynebacterium acetoacidophilumn 21476 Corynebacterium acetoacidophilumn 13870 Corynebacterium acetoglutam icum BI 11473 Corynebacterium acetoglutamnicumn BI 11475 Corynebacterium acetoglutam icumn 15806 Corynebacterium acetoglutamnicumn 21491 Corynebacterium acetoglutamnicumn 31270 Corynebacterium acetophilumn B3671 Corynebacterium am moniagenes 6872 2399 Corynebacterium arnmoniagenes 15511 Corynebaccerium fujiokense 21496 Corynebacterium glutamnicumn 14067 Corynebacterium glutamnicumn 39137 Corynebacterium glutamicum 21254 Corynebacterium glutamnicum 21255 Corynebacterium glutamnicumn 31830 Corynebacterium glutamnicumn 13032 Corynebacterium glutamnicumn 14305 Coryhnebacterium glutamnicumn 15455 Corynebacterium glutamnicumn 13058 Corynebacterium glutamnicumn 13059 Corynebacterium glutamnicumn 13060 Corynebacterium glutamnicumn 21492 Corynebacterium glutamnicumn Corynebacterium glutamnicumn 21526 Corynebacterium glutamnicumn 21543 Corynebacterium glutamnicumn Corynebacterium glutarnicumn 21851 Corynebacterium glutamnicumn 21253 Corynebacterium glutarnicurn 21514 Corynebacterium Iglutamicum 21516 Corynebacterium Iglutamnicumn 21299

CA

Corynebacterium glutaniicum 21300 Corynebacterium glutarnicum 39684 Corynebacterium glutamicum 21488 Corynebacterium glutarnicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 19223 Corynebacterium glutamicum 13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum 21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum 21355 Corynebacteriumn glutamicum 31808 Gorynebacterium glutamicum 21674 Corynebacterium glutainicum 21562 Corynebacterium glutamicum 21563 Corynebacterium glutamicum 21564 Corynebacterium giutamicum 21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum 21567 Corynebacterium glutamicum 21568 Corynebacteriurn glutam icum 21569 Corynebacterium glutamnicum 21570 Corynebacterium glutarnicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutaniicum- 21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum 19051 Corynebacterium glutamicum 19052 Coi-ynebacterium glutamicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutam icum 19056 Corynebacterium glutam icum 19057 Corynebacterium ltmcm108 Corynebacterium guaicm109___ Corynebacterium glutamicum 19060 Corynebacterium giutamicum 1328059__ Coryncbacterium glutamicum 151060____ Corynebacterium glutamicum 19157___ Corynebacterium glutam icum 1284 Corynebacterium glutainicum 21492 Corynebacterium glutamicum 21527 Corynebacterium glutamicum 8182 Cor-ynebacteriurn glutamicum 812416 Corynebacterium glutam icum B 12417 89 Ginu SIT N4B, L-'EC ,EE~ FNRJ gEj1 VCIMB §CSN T .S Corynebacterium glutamicum B 12418 Corynebacterium glutamicum BI 1476 Corynebacterium glutamicum 21608 Corynebacteriumn lilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacteriumn spec. 31088 Corynebacteriumn spec. 31089 Corynebacterium spec. 3 1090 Corynebacterium spec. 31090 Corynebacteriumn 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, Baarn, NL NCTC: National Collection of Type Cultures, London, UK DSMZ: Deutsche Sammlung von Mikroorganismen und Zelikulturen, Braunschweig, Germany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (40'edn), World federation for culture collections world data center on microorganisms, Saimnata, Japen.

2007203042 29 Jun 2007 Table 4: Alignment Results Length Accession Name of Genbank Hi1 ID ft length Genbank Hit (NTl rxaOO03 996 GB-GSS4:A0713475 581 AQ713475 GBHTG3:AC007420 GBHTG3:AC007420 GBBA1:MTCY3A2 1130583 AC007420 130583 AC007420 25830 Z83867 HS_5402_B?_Al2_*T7A RPCI-l 1 Human Male BAG Library Homo sapiens genomic clone Plate=978 Col=24 Row=B, genomic survey sequence.

Drosophila melanogaster chromosome 2 clone BACRO7M1O0(D630) RPCI-98 07.M. 10 map 24A-24D strain y; cn bw sp, -SEQUENCING IN PROGRESS -,83 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR07MIO0 (D3630) RPCI-98 07.M.10 map 24A-24D strain y: cn bw sp. ~SEQUENCING IN PROGRESS-, 83 unordered pieces.

Mycobacteriumn tuberculosis H37Rv complete genome; segment 136/162.

Source of Genbank Hit Homo sapiens Drosophila metanogaster Drosophila metanogaster %/homologiy Date of (GAP) Deposlt 37.148 13-Jul-99 34,568 34.568 58,140 rxaOO04 903 Mycobacterium GBBAI:MLCB1779 GBBA1:SAPURCLUS rxaOOO3O 513 GBEST21:C89713 GB-EST28:A1497294 GBEST21:C92 167 43254 9 120 767 Z98271 X92429 C89713 484 A1497294 637 C92167 189370 AF010496 tuberculosis Mycobacteriumn Ieprae cosmid B 1779. Mycobacterium leprae 57.589 S.alboniger napH, pur7, purlO, pur6, pur4, pur5 and pur3 genes. Streptomyces anulatus 55.667 C89713 Dictyostetium discoideum SS (H.Urushihara) Dictyosteliumn discoideum Dictyostelium discoideumn 45.283 cDNA clone SSG229, mRNA sequence.

tb63g03.yl Zebrafish WashU MPIMIG EST Danio rerio cDNA 5' similar to Danio rerio 42,991 SW:AFP4_MYOOC P80961 ANTIFREEZE PROTEIN LS-12. mRNA sequence.

C92167 Dictyoslelium discoideum SS (H.Urushihara) Dictyosteliumn discoideum Dictyostelium discoideum 44,444 cDNA clone SSD179, mRNA sequence.

Rhodobacter capsulatus strain S81003, partial genome. Rhodobacter capsulatus 39.689 20-Sep-99 20-Sep-99 17-Jun-98 8-Aug-97 28-Feb-96 20-Apr-98 1 1-MAR-1999 12-Jul-99 12-MAY-i1998 22-OCT-i1997 1 6-Jul-98 rxaO32 1632 GB-BA2:AFO1 0496 GBBA2:AF018073 GBBA2:AF045245 EM-PAT:E1 1760 GBPAT:126124 GB-INI1:LMFL5883 EM_PAT:E 11760 9810 AF018073 Rhodobacter sphaeroides operon regulator (smoG), perlplasmic sorbitol-binding Rhodobacter sphaeroides 48,045 protein (smoE), sortbitol/mannitol transport inner membrane protein (smoF), sortbitoilmannitol transport inner membrane protein (smoG), sorbitol/mannitol transport ATP-blndtng transport protein (smoK), sorbitol dehydrogenase (smoS), mannitol dehydrogenase (mIK), and periplasmic mannitol-binding protein (smoM) genes, complete cds.

5930 AF045245 Kiebsielta pneumoniae D-arabinitol transporter (daIT), D-arabinitot kinase Klebsiella pneumoniae 38,514 (dalK), D-arabinitol dehydrogenase (daiD)), and repressor (daIR) genes.

complete cds.

6911 El11760 Base sequence of sucrase gene. Corynebacterium 99,031 rxaOO41 1342 08-OCT-I1997 glutamicum 6911 126124 Sequence 4 from patent US 5556776. Unknown.

31934 ALI117384 Leishmania major Frledlin chromosome 23 cosmid L5883, complete sequence. Leishmania major Created) 99,031 07-OCT-1996 43,663 21-OCT-1999 94,767 08-OCT-I1997 (Rel. 52, Created) 94,767 07-OCT- 1996 rxaO42 882 6911 E1 1760 Base sequence of sucrase gene.

Corynebacteriumn glutamicum GB-PAT:126124 6911 126124 Seuce4fopaetU5567.nnwn Sequence 4 from patent US 5556776.

Unknown.

2007203042 29 Jun 2007 GBINI:CEU33051 rxa00043 1287 GB PAT:126124 EMPAT:E1 1760 GBPR3;AC005174 rxa00098 1743 GB-BA1:MSU88433 GBBA1:SC5A7 G8-BA1:MTCY10D7 ra00148 2334 GBBA1:MTCY277 GB-BA1 :MSGY456 GB-BA1:MSGY1 75 rxaOOl49 1971 GB-BA1:MSGY456 GBBA1:MSGY175 GBBA1:MTCY277 684 GBBA1:MTCY274 GBBAI:MSGB1529CS GBBA1:MTCY274 rxa00196 738 GBBA1:MTCY274 GBBA1:MTCY274

GBRO:RATCBRQ

rxa00202 1065 GB-ESTI:AA253618 GBEST26:A1390284 4899 6911 6911 39769 1928 40337 39800 38300 37316 18106 37316 18106 38300 39991 36985 39991 39991 39991 10752 313 490 Table 4 (continued) U33051 Caenorhabditis elegans sur-2 mRNA, complete cds.

126124 Sequence 4 from patent US 5556776.

Caenorhabditis elegans Unknown.

40,276 23-Jan-96 El 11760 Base sequence of sucrase gene. Corynebacteriumn glutamnicum AC005174 Homo sapiens clone UWGC:g1564a012 from 7p14-15, complete sequence. Homo sapiens U88433 Mycobacteriumn smegmatis phosphoglucose isomerase gene, complete cds. Mycobacteriumn smegmatis AL031 107 Streptomyces coelicolor cosmid 5A7. Streptomyces coeticolor Z79700 Mycobacterlum tuberculosis H37Rv complete genome; segment 44/162. Mycobacterium tuberculosis Z79701 Mycobacterium tuberculosis H37Rv complete genome; segment 65/1 62. Mycobacteriumn tuberculosis ADOOQOOlI Mycobacterium tuberculosis sequence from clone y456. Mycobacteriumn tuberculosis AD000015 Mycobacterium tuberculosis sequence from clone y1 75. Mycobacteriumn tuberculosis ADOQOOQOI Mycobacteriumn tuberculosis sequence from clone y456. Mycobacterium tuberculosis AD000015 Mycobacteriumn tuberculosis sequence from clone y175. Mycobacterumn tuberculosis Z79701 Mycobacteriumn tuberculosis H37Rv complete genome; segment 65/162. Mycobacterium tuberculosis Z74024 Mycobacteriumn tuberculosis H37Rv complete genome; segment 126/1 62. Mycobacterium tuberculosis L78824 Mycobacterium leprae cosmid B 1529 DNA sequence. Mycobacteriumn leprae Z74024 Mycobacleriumn tuberculosis H37Rv complete genome: segment 126/162. Mycobacternum tuberculosis Z74024 Mycobacteriumn tuberculosis H37Rv complete genome: segment 126/162. Mycobacteriumn tuberculosis Z74024 Mycobacteriumn tuberculosis H37Rv complete genome; segment 126/I162. Mycobacterium tuberculosis M55532 Rat carbohydrate binding receptor gene, complete cds. Rattus norvegicus AA253618 mw95clO.rl Soares mouse NMVL Mus musculus cDNA clone IMAGE:678450 Mus musculus mRNA sequence.

A1390284 mw96a03.yI Soares mouse NMVL Mus musculus cONA clone IMAGE:678508 5' Mus musculus similar to TR:0091 71 009171 BETAINE-HOMOCYSTEINE METHYLTRANSFERASE:. mRNA sequence.

A1390280 mw9SclO.yl Scares mouse NMVL Mus musculus cONA clone IMAGE:678450 Mus musculus mRNA sequence.

97,591 07-OCT-1 996 97,591 08-OCT-i1997 (Ret. 52, Created) 35,879 24-Jun-98 62,658 19-Apr-97 37,638 27-Jul-98 36,784 17-Jun-98 67.457 17-Jun-98 4 0,883 03-DEC-i1996 67,457 10-DEC-1996 35,883 03-DEC-1996 (0 51,001 10-DEC-1996 51,001 17-Jun-98 35,735 19-Jun-98 57,014 15-Jun-96 41,892 19-Jun-98 41,841 19-Jun-98 36.599 19-Jun-98 36.212 27-Apr-93 38,816 13-MAR-1997 42,239 2-Feb-99 37,307 2-Feb-99 58,312 17-Sep-97 36,632 23-Jun-99 GBEST26:A1390280 467 rxa00206 1161 GB-BA1 :MLCB637 44882 Z99263 Mycobacterium leprae cosmid B637.

GB-BA :MIVO1 2 70287 AL-021287 Mycobacterlum tuberculosis H37Rv complete genome; segment 132/162.

Mycobacterium leprae Mycobacterium tuberculosis 2007203042 29 Jun 2007 GB_BA1:SC6EI0 rxa00224 1074 GBBAi:6JU32230 GB_BA1:PDEETFAB GBHTG3:AC009689 rxa00225 909 GBRO:AF060178 GBGSS I1:A0325043 GBEST31:Al6764i3 rxa00235 1398 GB_BA1:MTCYIOG2 GBBA2:AF061753 GBBA2:AF086791 rxa00246 1158 GBBA2:AF012550 GBPAT:E03856 GBBA1 :BACADHT rxa0O251 831 GB-BA1:MTCY2OG9 GBBA1:MTVOO4 GBBA1:M1VOO4 rxaCO288 1134 GB-BA2:AF050114 GBGSS3:B16984 GBJIN2:AF144549 rxa00293 1035 GB-ESTI:T28483 23990 1769 2440 177954 2057 734 551 38970 3721 37867 2690 1506 1688 37218 69350 69350 1038 469 7887 313 AL1 09661 U32230 Li14864 AC009689 AF060178 A0325043 A1676413 Z92539 AF061 753 AF086791 AF01 2550 E03856 D90421 Z77 162 AL0091 98 AL0091 98 AF0501 14 B816984 AF144549 T28483 Table 4 (continued) Streptomyces coelicolor cosmid 6E10. Streptomyces coelicolor 38,616 A3(2) Bradyrhizobium japonicumn electron transfer flavoprotein small subunit (effS) nd Bradyrhizobium japonicum 48,038 large subunit (etft) genes, complete cds.

Paracoccus denitrificans electron transfer flavoprotein alpha and beta subunit Paracoccus denitrificans 48,351 genes, complete cdlss.

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

SAMPLING.

Mus musculus heparan sulfate 2-sulfotransferase (Hs2st) mRNA, complete cds.Mus musculus 39,506 mgxbOO2OJ0lr CUGI Rice Blast BAC Library Magnaporthe grisea genomic Magnaporthe grisea 38.333 clone mgxbOO20JOlir. genomic survey sequence.

etmEST0l67 EtHi Elmeria tenella cDNA clone elmc074 mRNA sequence. Eimeria tenella 35,542 Mycobacteriumn tuberculosis H37Rv complete genome: segment 471162. Mycobacterium 65,759 tuberculosis Nitrosomonas europaea CTP synthase (pyrG) gene, partial cds; and enolase Nitrosomonas europaea 58,941 (eno) gene, complete cdls.

Zymomonas mobllis strain ZM4 clone 67E 10 carbamnoylphosphale synthetase Zymomonas mobilis 61,239 small subunit (carA), carbamnoylphosphate 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. BD413 ComP (comP) gene, complete cds. Acinetobacter sp. BD413 53,726 gDNA encoding alcohol dlehydrogenase. Bacillus 51,688 stearothermophilus B.slearothermophilus adhT gene for alcohol dlehydrogenase. Bacillus 51,602 stearotherrnophilus Mycobacterium tuberculosis H37RV complete genome; segment 25/1 62. Mycobacterlumn 42,875 tuberculosis Mycobacteriumn tuberculosis H37Rv complete genome; segment 144/162. Mycobacteriumn 40,380 tuberculosis Mycobacterium tuberculosis H37Rv complete genome: segment 144/162. Mycobacteriumn 41,789 tuberculosis Pseudomonas sp. W7 alginate lyase gene, complete cdls. Pseudomonas sp. Wi7 49,898 344A14.TVC CIT978SKAI Homo sapiens genomic clone A-344A14, genomic Homo sapiens 39,355 survey sequence.

Aedes albopictus ribosomal protein L34 (rp134) gene, complete cds. Aedes albopictus 36,509 EST46 182 Human Kidney Homo sapiens cDNA 3Tend similar to flavin- Homo sapiens 42,997 containing monooxygenase 1 (H-T:1956), mRNA sequence.

5-Aug-99 25-MAY-i1996 27-OCT-1993 28-Aug-99 18-Jun-98 8-Jan-99 19-MAY-i1999 17-Jun-98 31-Aug-98 4-Nov-95 27-Sep-99 29-Sep-97 7-Feb-99 17-Jun-98 18-Jun-98 18-Jun-98 03-MAR-i1999 4-Jun-98 3-Jun-99 6-Sep-95 2007203042 29 Jun 2007 GB_-PRI:HUMFMO1 2134 M64082 GOBEST32:A734238 512 A1734238 rxa00296 2967 GBHTG6:ACO1 1069 168266 AC011069 GB-EST5:AA531468 GBHTG6:AC01 1069 rxa00310 558 GBVI:VMVY1678O

GBVI:VARCG

GB VI: WCGAA rxaOO317 777 GBHTG3:AC009571 GB-HTG3:AC009571 GBPR3:AC005697 rxa00327 507 GBBAI:LCATPASEB GB-BA1:LCATPASEB rxa00328 615 GBBA1:STYPUTPE GB_BA1:STYPUTPF GB-BA1 :STYPUTPI rxa00329 1347 GBPR3:AC004691 GBPR4.AC004916 GBPR3:AC004691 rxa00340 1269 GBBA1:MTCY427 GB-GSS1 2:AQ4 12290 GB_PL2:AF1 12871 rxa00379 307 GB-HTGi:CEY56A3 GBHTG1:CEYS6A3 414 168266 '186986 186103 185578 159648 159648 174503 1514 1514 1887 1887 1889 141990 129014 141990 38110 238 2394 224746 224746 AA53 1468 AC01 1069 Y16780 L22579 X69198 AC009571 AC009571 AC005697 X64542 X64542 L01 138 L01 139 L01 142 AC004691 AC004916 AC004691 Z70692 AQ4 12290 AF112871 AL022280 AL022280 Table 4 (continued) Human flavin-containing monoaxygenase (FMO1) mRNA, complete cds.

zb73c05.y5 Soares~jetal-lungNbHL19W Homo sapiens cONA clone IMAGE:309224 5' similar to gb:M64082 DIMETHYLANILINE MONOOXYGENASE (HUMAN);, mRNA sequence.

Drosophila melanogaster chromosome X clone BACRI 1 H20 (D881) RPCI-98 I 1.H.20 map 1213-1 2C strain y; cn bw sp, SEQUENCING IN PROGRESS unordered pieces.

nj63d12.sl NCICGAP PrlO Homo sapiens cDNA clone IMAGE:997175, mRNA sequence.

Drosophila melanogaster chromosome X clone BACRI I1H20 (0881) RPCI-98 I11.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 4 clone 57 A 22 map 4, SEQUENCING IN PROGRESS 8 unordered pieces.

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

L.casei gene for ATPase beta-subunit.

L.casei gene for ATPase beta-subunit.

Salmonella (52980) proline permease (putP) gene, 5' end.

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

Salmonella (S3015) proline permease (pulP) gene. 5Tend.

Homo sapiens PAC clone DJ0740D02 from 7p14-p15, complete sequence.

Homo sapiens clone DJ0891 L14, complete sequence.

Homo sapiens FAG clone DJ0740D02 from 7p14-plS, complete sequence.

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

RPCI-1 1-195H2.TV RPCI-1 1 Homo sapiens genomic clone RPCI-1 1-195H2.

genomlc survey sequence.

Astasia longa small subunit ribosomal RNA gene, complete sequence.

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

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

Drosophila melanogaster 33,890 Homo sapiens Homo sapiens 37.915 8-Nov-94 41,502 14-Jun-99 Homo sapiens Drosophila metanogaster variola minor virus Variola major virus Variola virus Homo sapiens Homo sapiens Homo sapiens Lactobacillus case Lactobacillus case! Salmonella sp.

Salmonella sp.

Salmonella sp.

Homo sapiens Homo sapiens Homo sapiens Mycobacterium tuberculosis Homo sapiens Aslasia longa Caenorhabditis elegans Caenorhabditis elegans 40,821 30.963 35,883 34.664 36.000 36.988 36,988 36,340 34.64 39.308 39.623 39,623 42,906 38.142 38,549 35,865 38,940 36,555 36.465 35,179 35,179 02-DEC-i 999 20-Aug-97 02-DEC-i1999 2-Sep-99 12-Jan-95 13-DEC-i1996 29-Sep-99 29-Sep-99 09-OCT-i1998 11 -DEC-i 992 1 1-DEC-1992 09-MAY-I1996 09-MAY-1996 09-MAY-i1996 1 6-MAY-I1998 17-Jul-99 16-MAY-i1998 24-Jun-99 23-MAR- 1999 28-Jun-99 6-Sep-99 6-Sep-99 2007203042 29 Jun 2007 GBPR2:HS134019 GBGSS4:AQ730532 86897 416 rxaGO381 729 Table 4 (continued) AL034555 Human DNA sequence from clone 134019 on chromosome lp36.1 1-36.33. Homo sapiens complete sequence.

AQ730532 HS_2149_Al_COS_T7C CIT Approved Human Genomic Sperm Library D Homo sapiens Homo sapiens genomic clone Plate=2149 001=1 1 Row--E, genomic survey sequence.

A1120939 ub74f05.rl Scares mouse mammary gland NMLMG Mus musculus cDNA clone Mus musculus IMAGE:1383489 5' slmilar to gb:J04046 CALMODULIN (HUMAN): gb:M19381 Mouse calmodulin (MOUSE);, mRNA sequence.

A1120939 ub74f05.rl Scares mouse mammary gland NMLMG Mus musculus cDNA clone Mus musculus IMAGE:1383489 5'sImilar to gb:J04046 CALMODULIN (HUMAN); gb:MI93Bi 40.604 35.766 23-Nov-99 1 5-Jul-99 GBE5T23:A1120939 561 41,113 2-Sep-98 41,113 2-Sep-98 GBE5T23:A1120939 GBE5T32:A1726450 GBG554:A0740856 rxaOO385 362 GB_PR1:HSPAIP rxa00388 1134 GBBA1:MTY25D1O GB-BAI:MSGY224 GBHTG1:AP000471 rxa00427 909 GB BA1:MSGY126 GBBA1:MTY13DI2 GBHTG1:CEY48C3 rxa00483 1587 GB_PR2:HSAFOOI155O GBBAl :LLCPJW565 GB_HTG2:AC0067541 nxa00511 615 GB_PR3:HSE127C11 GB_PR3:HSE127C11 rxaOO512 718 GB-BA1:MTCY22G8 561 565 768 1587 40838 40051 72466 37 164 37085 270193 173882 12828 206217 38423 38423 22550 A1726450 AQ740856 X91809 Z95558 AD000004 AP000471 AD00001 2 Z80343 Z92855 AF001 550 Y12736 AC006754 Z74581 Z74 581 Z95585 Mouse calmoadulin (MOUSE);, mRNA sequence.

BNLGHi5857 Six-day Cotton fiber Gossypium hirsutum cDNA 5' similar to (AF01 5913) Skbl Hs (Home sapiens). mRNA sequence.

HS_2274_A2_A07_170 CIT Approved Human Genomic Sperm Library D Homo sapiens genomic clone Plate=2274 001=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 y22 4 Homo sapiens chromosome 21 clone B2308H-15 map 21q22.3, SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterium tuberculosis sequence from clone y126.

Mycobacterium tuberculosis H-37Rv complete genome: segment 156/162.

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

Homo sapiens chromosome 16 BAC clone CIT987SK-33401 1 complete sequence.

Lactococcus ladtis cremoris plasmid pJW565 DNA, abiiM, abiiR genes and orfX.

Caenorhabditis etegans clone Y4OB1O, -SEQUENCING IN PROGRESS unordered pieces.

Human DNA sequence from cosmid E127CI I on chromosome 22q1 1.2-qter contains STS.

Human DNA sequence from cosmid El 27011I on chromosome 22q1 1 .2-qter contains STS.

Mycobacterium tuberculosis H37Rv complete genome: segment 49/162.

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

cremoris Caenorhabditis elegans Homo sapiens Home sapiens 41,152 41,360 36,792 51,852 51,852 36,875 60.022 60.022 28,013 38,226 37,492 36,648 39,831 36.409 56,232 11-Jun-99 16-Jul-99 29-MAR-i1996 17-Jun-98 03-DEC-i1996 13-Sep-99 10-DEC-1996 17-Jun-98 29-MAY-i1999 22-Aug-97 01-MAR-i1999 23-Feb-99 23-Nov-99 23-Nov-99 17-Jun-98 Mycobacterlum tuberculosis 2007203042 29 Jun 2007 Table 4 (continued) M.smegmatis gItA gene for citrate synthase.

GBBA1:MSGLTA 1776 X60513 Mycobacterium smegmatis 56.143 20-Sep-91 rxaOO517 1164 GB-ITG2:AC006911I GBHTG2:AC006911 GBE5T29:A1602158 rxaOOSIB 320 GBBA2:ECU73857 GBBA2:STU51879 rxa00606 2378 1860 GB BA2:AE000140 GBEST32:AU068253 GBEST13:AA363046 GB-EST32:AU068253 GBBA1:PAORF1 GBBA1:PAORF1 128824 298804 298804 481 128824 8371 12498 376 329 376 1440 1440 80381 81493 80381 38400 81493 43481 197110 197110 181745 U73857 AC006911 AC00691 1 A16021 58 U73857 U51879 AE000 140 AU068253 AA363046 AU068253 X13378 X1 3378 AC010871 X98 130 AC01 0871 AC004058 X98130 AB026648 AC01 0325 AC01 0325 AC008 179 Escherichia coli chromosome minutes 6-8.

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

Caenorhabditls elegans clone Y94H6x, SEQUENCING IN PROGRESS ,Caenorhabdilis elegans unordered pieces.

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

Escherichia col chromosome minutes 6-8. Escherichia col Salmonella typhimurium propionate catabolism operon: RpoN activator protein Salmonella typhimurium homolog (prpR), carboxyphosphonoenolpyruvate phosphonomutase homolog (prpB), citrate synthase homolog (prpC), prpD and prpE genes, complete cds.

Escherichla coli K-12 MG1655 section 30 of 400 of the complete genome. Escherichia coi AU068253 Rice callus Oryza sativa cDNA clone 012658_9A, mRNA sequence. Oryza saliva EST72922 Ovary 11 Homo sapiens cDNA 5'end, mRNA sequence. Homo sapiens AU068253 Rice callus Oryza saliva cONA clone 012658_9A, mRNA sequence. Oryza saliva Pseudomonas amyloderamosa DNA for ORF 1. Pseudomonas amyloderamosa Pseudomonas amyloderamosa DNA for ORF I. Pseudomonas amyloderamosa Arabidopsis thaliana chromosome III BAC T16011 genomic sequence, Arabidopsis thaliana complete sequence.

A.thaliana 81kb genomc sequence. Arabidopsis thaliana Arabidopsis thaliana chromosome IIl BAC T16011 genomic sequence, Arabidopsis thaliana complete sequence.

Homo sapiens chromosome 4 clone 6241 P1 9 map 4q25. complete sequence. Homo sapiens A.thaliana 81kb genomic sequence. Arabidopsis thaliana Arabidopsis thaliana genomic DNA, chromosome 3, P1 clone: MIJiS5. complete Arabidopsls thaliana sequence.

Homo sapiens chromosome 19 clone CITB-El-256BA7,~ SEQUENCING IN Homo sapiens PROGRESS 40 unordered pieces.

Homo sapiens chromosome 19 clone ClTB-E1-2568A1 7, SEQUENCING IN Homo sapiens PROGRESS 40 unordered pieces.

Homo sapiens clone NH0576F0i, 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 1 2-Nov-98 7-Jun-99 21-Apr-97 7-Jun-99 14-Jul-95 14-Jul-95 Escherichia coli 48.563 14-Jul-99 rxa00679 1389 GBPL2:AC010871 GB-PL1:AT8IKBGEN GB-PL2:AC010871 rxaOO6BO 441 GBPR3:AC004058 GBPL1:AT81KBGEN GBPL1:AB026648 nxa00682 2022 GB_-TG3:AC010325 GB-HTG3:ACO1 0325 GBPR4:AC008179 13-Nov-99 12-MAR-I 997 13-Nov-99 30-Sep-98 1 2-MAR-i1997 07-MAY-i1999 15-Sep-99 15-Sep-99 28-Sep-99 2007203042 29 Jun 2007 rxa00683 1215 GB-BA2:AE000896 10707 GBINi:DMBR7A4 212734 GBEST35:AV163010 273 rxa00686 927 GBHTG2:HSDJI37K2 190223 GBHTG2:HSDJ137K2 190223 GB_ESTI12:MA284399 431 rxaO00D 927 GBEST34:A1785570 454 GB-EST25:A1256147 684 Table 4 (continued) AE000896 Methanobacterium therrnoautotrophicumn from bases 1189349 to 1200055 (section 102 of 148) of the complete genome.

AL109630 Drosophila melanogaster clone BACR7A4.

AV163010 AV163010 Mus musculus head 05781J6J 13-day embryo Mus musculus cONA clone 3110006J22. mRNA sequence.

AL049820 Homo sapiens chromosome 6 clone RP1-1 371<2 map q25.1-25.3, SEQUENCING IN PROGRESS In unordered pieces.

AL049820 Homo sapiens chromosome 6 clone RP1-137K2 map q25.1-25.3.

SEQUENCING IN PROGRESS -,in unordered pieces.

AA284399 zs57b04.rl NCI_OGAP_GCB1 Homo sapiens cDNA clone IMAGE:701551 5., mRNA sequence- A1785570 uj44d03.xl Sugano mouse liver mla Mus musculus cDNA clone IMAGE:1 922789 3' similar to gb:Z28407 60S RIBOSOMAL PROTEIN L8 (HUMAN);, mRNA sequence.

A1256147 ui95el2.xl Sugano mouse liver mlia Mus musculus cDNA clone IMAGE:1890190 3' sImilar to gb:Z28407 603 RIBOSOMAL PROTEIN 18 (HUMAN):, mRNA sequence.

X14979 C. aurantiacus reaction center genes 1 and 2.

AL109732 Streptomyces coelicolor cosmid 71-2.

Z74024 Mycobacterium tuberculosis H37Rv complete genome; segment 126/162.

Methanobacterium thermoautolrophicum Drosophila melanogaster Mus musculus Homo sapiens Homo sapiens Homo sapiens Mus musculus Mus musculus Chloroflexus aurantlacus Streptomyces coelicolor A3(2) Mycobacterium tuberculosis rxaOO703 2409 rxaOO705 1038 rxa00782 1005 rxa00783 1395 ra00794 1128 GBBA1:CARCG12 GBBA1:5C7H2 GBBAi:MTCY274 GBBA2:REU60056 GB-GSS1 5:A0604477 2079 42655 39991 2520 505 GB_EST1li:AA224340 443 GB-EST5:N30648 291 GB BAI:MTCY1007 39800 GBBA1:MLCL373 37304 GB-BA2:AF1 28399 2842 GBHTG2ACOO8 158 118792 GBHTG2:AC008158 118792 GSBPR3:AC005017 137176 GB-BA1:MTVO17 67200 U60056 A0604477 AA224340 N30W4 Z79700 AL035500 AF128399 AC008158 AC008158 AC00501 7 AL02 1897 38,429 36,454 41,758 38,031 38,031 39,205 4 1,943 40,791 37.721 56,646 37,369 5 1,087 39,617 35,129 43,986 63,327 62.300 53,698 35.135 35,135 35,864 40.331 Ralstonia eutropha formate de hyd roge nase -like protein (cbb~c) gene, complete Ralstonia eutropha cds.

HS_-2116_Bi_G07_MR ClT Approved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomlc clone Plale=2116 001=1 3 Row=N, genomic survey sequence.

zrI4eO7.sl1 Stratagene hNT neuron (#937233) Homo sapiens cDNA clone Homo sapiens IMAGE:648804 3% mRNA sequence.

yw77b02.sl Soares~placenta_-8o9weeks-2NbHP8o9W Homo sapiens cDNA Homo sapiens clone IMAGE:258219 mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 441162. Mycobacterium tuberculosis Mycobacterium leprae cosmid L373. Mycobacteriumn lepr Pseudomonas aeruginosa succlnyl-CoA synthetase beta subunit (sucC) and Pseudomonas aeru succinyl.CoA synthelase alpha subunit (sucD) genes, complete cds.

Homo sapiens chromosome 17 clone hRPK.42F20 map 17, Homo sapiens SEQUENCING IN PROGRESS 14 unordered pieces.

Homo sapiens chromosome 17 clone hRPK.42_F -20 map 17, Homo sapiens SEQUENCING IN PROGRESS 14 unordered pieces.

Homo sapiens BAG clone GS214N13 from 7 p14-p15, complete sequence. Homo sapiens Mycobacterium tuberculosis H37Rv complete genome; segment 48/162. Mycobacterium tuberculosis 1 5-Nov-97 30-Jul-99 8-Jul-99 03-DEC-1999 03-DEC.1999 14-Aug-97 2-Jul-99 12-Nlov-98 23-Apr-91 2-Aug-99 19-Jun-98 16-OCT-i1996 10-Jun-99 11 -MAR- 1998 5-Jan-96 17-Jun-98 27-Aug-99 25-MAR-i1999 28-Jul-99 28-Jul-99 8-Aug-98 24-Jun-99 ae ginosa 2007203042 29 Jun 2007 GB BAI:MLCB1222 34714 AL049491 GBPR2:HS15lBi4 128942 Z82188 rxa00799 1767 GBPL2:AF016327 616 AF016327 GBHTG2:HSDJ31 9M7 128208 AL079341 GBHTG2:HSDJ319M7 128208 AL079341 Table 4 (continued) Mycobacterium leprae cosmid B1222.

Human DNA sequence from clone 15181i4 on chromosome 22 Contains SOMATOSTATIN RECEPTOR TYPE 3 (SS3R) gene,pseudogene similar to ribosomal protein L39,RAC2 (RAS-RELATED C3 BOTULINUM TOXIN SUBSTRATE 2 (P21-RAC2)) gene ESTs, STSs, GSSs and CpG Istands.

complete sequence.

Hordeum vulgare Barpermi (permil) mRNA, partial cds.

Homo sapiens chromosome 6 clone RPi-31 9M7 map p 21 .1-21.3.

SEQUENCING IN PROGRESS in unordered pieces.

Homo sapiens chromosome 6 clone RP1-319M7 map p21.1-21.3, SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome: segment 100/162.

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

S.cerevisiae SFA and ARP genes.

Mycobacterium leprae Homo sapiens Hordeum vulgare Homo sapiens Homo sapiens Mycobacterium tuberculosis Streptomyces coellcolor 61,170 27-Aug-99 37,455 16-Jun-99 41,311 01-OCT-1997 36.845 30-Nov-99 36.845 30-Nov-99 rxaCO800 1227 GB.,BAI:MTVO22 GBBA1:AB019513 GBPL1:SCSFAARP GB_BAI:MT-Yi5C1O GBBA1:MLCB2548 GBBA2:AF169031 13025 AL021925 4417 AB01 9513 7008 X68020 63,101 4 1,312 17-Jun-98 13-Nov-98 29-Nov-94 Saccharomyces cerevisiae 36,288 rxa00825 1056 33050 Z95435 Mycobacterium tuberculosis H-37Rv complete genome; segment 154/162.

38916 AL023093 1141 AF169031 rxa00871 rxaOO872 1077 GB_IN1:CEF23HI2 GBHTG2:AC007263 GBHTG2:AC007263 GBBAi:MTVO49 35564 Z74472 167390 AC007263 167390 AC007263 40360 AL022021 Mycobacterlum ieprae cosmid 82548.

Xanthomonas oryzae pv. oryzae putative sugar nucleotide epimerase/dehydratase gene, partial cds.

Caenorhabditis elegans cosmid F23H 12. complete sequence.

Homo sapiens chromosome 14 clone BAG 79J20 map 14q31.

SEQUENCING IN PROGRESS 5 ordered pieces.

Homo sapiens chromosome 14 clone BAG 79,120 map 14q31, SEQUENCING IN PROGRESS 5 ordered pieces.

Mycobacterium tuberculosis H37Rv complete genome: segment 81/162.

Candida dubtiniensis ACTI gene, exons 1-2.

Candide albIcans acti gene for actin.

Rhodobacter capsulatus strain SB1003, partial genome.

Mycobacterium tuberculosis Mycobacterium leprae Xanthomonas oryzae pv.

oryzae Caenorhabditis elegans Homo sapiens Homo sapiens Mycobacterium tuberculosis Candida dubliniensis Candida atbicans Rhodobacter capsulatus Sinorhizobium meliloti Paralichthys otivaceus 34,502 08-OCT-1999 35,714 24-MAY-i1999 35.714 24-MAY-1999 36,981 19-Jun-98 39,435 46,232 27-Aug-99 14-Sep-99 39,980 17-Jun-98 rxaO0879 2241 GBPL2:CDU236897 GB PLI1:CAACTiA ncaOO9O9 955 GB-BA2:AF010496 1827 3206 189370 AJ236897 X16377 AF01 0496 38,716 36,610 51,586 1 -Sep-99 10O-Apr-93 12-MAY-i1998

GB_BAI:RMPHA

GBEST16:C23528 7888 X93358 Rhizobium melitoti pha[A,B,C.D.E,F,G] genes. 48,367 12-MAR-1999 41,640 28-Sep-99 317 C23528 C23528 Japanese flounder spleen Paralichlhys olivaceus cONA clone H65(2).

mRNA sequence.

Homo sapiens chromosome 18 clone hRPK.44_0_1 map 18, SEQUENCING IN PROGRESS-, 18 unordered pieces.

=0aO9Q3 2118 GB-HTG2:AC007734 188267 AC007734 Homo sapiens 34.457 5-Jun-99 2007203042 29 Jun 2007 rxa00945 1095 GB HTG2:AC007 734 GBEST18:MA709478 GBHTG4:AC010351 GBHTG4:ACOIO3S1 GBBA1:MTCYO5A6 188267 406 220710 220710 38631 AC007734 AA709478 AC0 10351 ACO 10351 Z96072 Table 4 (continued) Homo sapiens chromosome 18 clone hRPK.44 0 1 map 18, SEQUENCING IN PROGRESS 18 unordered pieces.

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

Homo sapiens chromosome 5 clone CITB-H1_202286. SEQUENCING IN PROGRESS 68 unordered pieces.

Homo sapiens chromosome 5 clone CITB-H-2022B6, SEQUENCING IN PROGRESS 68 unordered pieces.

Mycobacterlum tuberculosis H37Rv complete genome; segment 120/162.

Homo sapiens Mus musculus Homo sapiens Homo sapiens Mycobacterium tuberculosis 34.457 42,065 36,448 36,448 36.218 5-Jun-99 24-DEC-1 997 31-OCT-i1999 31-OCT-1999 17-Jun-98 rxa00965 rxa00999 1575 GBPAT:E13660 GBBAl :MTCY359 GBBA1:MLCB1788 rxaOl0lS 442 GBBA1:MTVOO8 GB-BA1 :MTVOO8 1916 36021 39228 63033 63033 rxaOlO2S 1119 GBBA1:SC7A1 32039 GBBA1:MSGB1723CS 38477 GBBA1:MLCB637 44882 rxa0lO48 1347 GBBA2:AF017444 3067 GBBA1;BSU80013 218470 GBVI:HSV2HG52 154746 rxa0lO4g 1605 GBHTG2:AC002518 131855 GBHTG2:AC00251 8 131855 GB-HTG2:AC00251 8 131855 rxa0 lO7l 1494 GBPR3:H5DJ653C5 85237 GB-BA1:ECU29579 72221 GBBA1:ECU29579 72221 rxa0 lO89 873 GBGSS8:AQ044021 387 E13660 Z83859 AL008609 AL021 246 AL021 246 AL034447 L78825 Z99263 AF0 17444 Z99 116 Z86099 ACD02518 AC00251 8 AC00251 8 AL0-49743 U29579 U29579 A004402 1 gONA encoding 6-phosphogluconate dehydrogenase.

Mycobacteriumn tuberculosis H37Rv complete genome; segment 84/162.

Mycobacterium leprae cosmid 8 1788.

Mycobacterium tuberculosis H37Rv complete genome; segment 108/162.

Mycobacterium tuberculosis H37Rv complete genome: segment 108/162.

Streptomyces coelicolor cosmid 7A1.

Mycobacteriumn leprae cosmid 81 723 DNA sequence.

Mycobacterium Ieprae cosmid 8637.

Sinorhizobium meliloli NADP-dependent malic enzyme (tine) gene, complete cds.

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

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

Homo sapiens chromosome X clone bWXD2O, -SEQUENCING IN PROGRESS 11I unordered pieces.

H-omo sapiens chromosome X clone bWXD2O, -SEQUENCING IN PROGRESS 111 unordered pieces.

Homo sapiens chromosome X clone bWXD2O, -SEQUENCING IN PROGRESS 11 unordered pieces.

Human DNA sequence from clone 653C5 on chromosome lp 2 l.3- 2 2.3 Contains CA repeat(D15435), STSs and GSSs, complete sequence.

Escherichla coi K-12 genome; approximately 611to62 minutes.

Escherichia coli K-12 genome; approximately 61 to 62 minutes.

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

Corynebacterium glutamicum Mycobacterium tuberculosis Mycobacterium teprae Mycobacterium tuberculosis Mycobacterium tuberculosis Streptomyces coeticolor Mycobacterium leprae Mycobacterium leprae Sinorhizobium meliloti Bacillus subtilis human herpesvirus 2 Homo sapiens Homo sapiens Homo sapiens Homo sapiens Escherichia coli Escherchla coli Homo sapiens 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 27-Aug-99 17-Jun-98 17-Jun-98 15-DEC-I1998 15-Jun-96 17-Sep-97 2-Nov-97 26-Nov-97 04-DEC-i 998 2-Sep-97 2-Sep-97 2-Sep-97 23-Nov-99 1-Jul-95 1-Jul-95 14-Jul-98 2007203042 29 Jun 2007 GBGSS8:A0042907 GBGSS8:AQ044021 rxa0iO93 1554 GB_BA1:CORPYKI GBBAI:MTCYO1B2 GBBA1:M1U65430 rxaOi099 948 GBBA2:AF045998 GBBA2:AF051846 GB_GSSi:FR0005503 rxa01lll1 541 GBPR3:AC004063 GBPR3:HS1 178121 GB-ITG3:AC009301 nxaOi130 687 GB-HTG3:AC009444 GBHTG3:AC009444 GBIN1 :OMC66A1 rxa0ll93 1572 GB_BAI:CGASO19 EMPAT:E09634 392 AQ042907 387 AQ044021 2795 L27126 35938 Z95554 1439 U65430 780 AF045998 738 AF051846 619 Z89313 '177014 AC004063 62268 AL109852 163369 AC009301 164587 AC009444 164587 AC009444 34127 AL031227 1452 X76875 1452 E09634 36241 U151186 1452 E09634 1452 X76875 414 X60570 Table 4 (continued) CIT-HSP-2318DI7.TR CIT-HSP Homo sapiens genomic clone 23181317. Homo sapiens genomic survey sequence.

CIT-HSP-231SC18.TR CIT-HSP Homo sapiens genomic clone 2318018, Homo sapiens genomic survey sequence.

Corynebacterium pyruvate kinase gene, complete cds. Corynebacterium glutamicum Mycobacterium tuberculosis H37Rv complete genome; segment 72/1 62. Mycobacterium tuberculosis Mycobacterium intracellulare pyruvale kinase (pykF) gene, complete cds. Mycobacterlumn intracellulare Corynebactenium glutamnicumn inositol monophosphate phosphatase (impA) Corynebacterium gene, complete cds. giutamicum Corynebacterium glutamicum phosphoribosyformimino-5-amino-1- Corynebacterium phosphoribosyl-4- Imidazolecarboxamide isomerase (hIsA) gene, complete giutamicum cds.

Frubripes GSS sequence, clone 079SBaE8, genomic survey sequence. Fugu rubripes Homo sapiens chromosome 4 clone 63218, complete sequence. Homo sapiens Human DNA sequence from clone RP5-1 178121 on chromosome X, complete Homo sapiens sequence.

Homo sapiens clone NH0062F1l4, -SEQUENCING IN PROGRESS 5 Homo sapiens unordered pieces.

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

Homo sapiens clone 1_0_3, SEQUENCING IN PROGRESS ,8 H-omo sapiens unordered pieces.

Drosophila melanogaster cosmid 66A1. Drosophila melanogaster C~glutamlcum (ASO 19) AlPase beta-subunit gene. Corynebacterlum glutamicum Brevibacleriumn flavumn UncO gene whose gene product is Involved in Corynebacterium glutamicum Mycobacterium leprae cosmid L471. Mycobacteriumn leprae Brevibacierium flavum UncD gene whose gene product is involved in Corynebacterium glutamicum C.glutamicum (ASO 19) ATPase beta-subunit gene. Corynebacterium glutamicum Hepatitis C genomic RNA for putative envelope protein (RE4B isolate). Hepatitis C virus 35,969 44 .54 5 100,000 63,771 67,061 99,615 100,000 37,785 35,835 37,873 37,240 38,416 38,416 38,416 99,931 99,242 39,153 100.000 100,000 36.769 14-Jul-98 14-Jul-98 07-DEC-1994 17-Jun-98 23-DEC-i1996 19-Feb-98 '12-MAR-i 998 01-MAR-1997 10-Jul-98 0 i-DEC-i 999 13-Aug-99 22-Aug-99 22-Aug-99 05-OCT-i1998 27-OCT-i1994 07-OCT-i 997 (Ret. 52, Created) 09-MAR-i1995 07-OCT-i997 (Rel. 52, Created) 27-OCT-i1994 5-Apr-92 rxa0l 194 495 GBBA1:MLU15186 EMPAT:E09634 GB8BA1:CGAS0i 9 GB_VI:HEPCRE4B rxaoi 200 2007203042 29 Jun 2007 rxa0 i2Oi 1764 GB-BA1:SLATPSYNA GBBA1:MTCY373 GB-BA1:MLU15186 rxa01202 1098 GB-BAI:SLATPSYNA GBBA1:SLATPSYNA GBBA1:MCSQSSHC rxa01204 933 GBPL1:AP000423 GBHTG6:AC009762 GB-ITG6:AC009762 rxa~l2l6 1124 GB-BAI:MTCYiOG2 GB-BA2:AF01 7435

GBBAI:CCRFLBDBA

rxa01225 1563 GBBA2:AF058302 GB-HTG3:AC007301 GBHTG3:AC007301 rxa01227 444 GBBA1:SERFDXA GBBA1:MTVO05 GBSAl :MSGY348 rxa0i242 900 GBPR3:AC005697 GBHTG3:ACO 10722 GB-HTG3:ACOI 0722 8560 35516 36241 8560 8560 5538 154478 '164070 164070 38970 4301 4424 25306 165741 Z22606 Z7341 9 U15 186 Z22606 Z22606 Y09978 AP000423 AC009762 AC009762 Z92539 AF0 17435 M69228 AF058302 AC007301 Table 4 (continued) Slividans i protein and ATP synthase genes. Streplomyces lividans 66,269 Mycobacterium tuberculosis I-37Rv complete genome; segment 57/1 62. Mycobacterium 65,437 tuberculosis Mycobacterium leprae cosmid L471. Mycobacterium teprae 39,302 Slividans i protein and ATP synthase genes. Streptomyces lividans 57,087 Slividans 1 protein and ATP synthase genes. Streptomyces lividans 38,298 M.capsulalus orfx, orfy. orfz, sqs and shc genes. Methylococcus capsulatus 37,626 Arabidopsis thaliana chioroplast genomic DNA. complete sequence, Chloroptast Arabidopsis 38,395 strain: Columbia. thaliana Homo sapiens clone RPI 1-114116. SEQUENCING IN PROGRESS *,39 H-omo sapiens 35,459 unordered pieces.

Homo sapiens clone RP1 1-114116. SEQUENCING IN PROGRESS *,39 Homo sapiens 36,117 unordered pieces.

Mycobacterlum tuberculosis H-37Rv complete genome; segment 471162. Mycobacterium 39.064 tuberculosis Methytobacterium extorquens methanol oxidation genes. gimU-Iike gene. Methylobacterium 42,671 partial cds, and orfL2, orfli, orfR genes, complete cds. extorquens Ocrescentus flagetlar gene promoter region. Caulobacter crescentus 41,054 Streptomyces roseofuivus frenolicin biosynthetic gone dluster, complete Streptomyces roseofulvus 36,205 sequence.

Drosophila melanogaster chromosome 2 clone BACRO4B09 (13576) RPCI-98 Drosophila melanogaster 39,922 04.B.9 map 43E1 2-44F1 strain y; cn bw sp, -SEQUENCING IN PROGRESS **,150 unordered pieces.

Drosophila meianogaster chromosome 2 clone BACRO4BO09 (D576) RPCI-98 Drosophila melanogaster 39,922 04.B.9 map 43E12-44F1 strain y; cn bw sp, SEQUENCING IN PROGRESS 150 unordered pieces.

Saccharopolyspora erythraea ferredoxin (fdxA) gene, complete cds. Saccharopolyspora 64,908 erythraea Mycobacteriumn tuberculosis H37Rv complete genome; segment 51/162. Mycobacterium 62,838 tuberculosis Mycobacteriumn tuberculosis sequence from clone y348. Mycobacterium 61,712 tuberculosis Homo sapiens chromosome 17. cione hRPK.138P22, complete sequence. Homo sapiens 35,373 Homo sapiens clone NH0122L-09, SEQUENCING IN PROGRESS Homo sapiens 39,863 unordered pieces.

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

01-MAY-1995 17-Jun-98 09-MAR-i1995 01-MAY-i1995 01-MAY- 1995 26-MAY-i1998 1 5-Sep-99 04-DEC-i1999 04-DEC-i1999 17-Jun-98 10-MAR-1g98 26-Apr-93 2-Jun-98 17-Aug-99 17-Aug-99 13-MAR-i1996 17-Jun-98 10-DEC-1996 09-OCT-i1998 25-Sep-99 25-Sep-99 '165741 AC007301 3869 37840 40056 174503 160723 M61119 AL010186 AD000020 ACD05697 AC010722 160723 AC010722 2007203042 29 Jun 2007 rxa01243 1083 GB-GSS1O:A0255057 GB-IN1:CEK05D4 GBIN1:CEK05D4 rxa0l259 981 GB-BA1:CGLPD GBHTG4:AC010567 GBI-ITG4:AC01 0567 rxa01262 1284 GBBA2:AF1 72324 GBBA2:ECU78086 GBBA1:090841 rxa01311 870 GBPR3:AG004103 GBHTG3:AC007383 GB-HTG3:AC007383 rna01312 2142 GB-BA2:AE000487 GB-BA1 :M1V016 GB-BA1 :U00022 rxa0l325 795 GBHTG4:AC009245 GB-HTG4:AG009245 GBHTG4.AC009245 rxa01332 576 GBHTG6:AC007186 GBHTG6:AG007147 583 19000 19000 1800 143287 143287 14263 A0255057 Z92804 Z92804 Y16642 AC0 10567 AC01 0567 AF1 72324 Table 4 (continued) mgxbGOO6N01 r CUGI Rice Blast BAG Library Magnaporthe grisea genomic Magnaporthe grisea clone mgxbOOOBN0l r, genomic survey sequence.

Caenorhabditis elegans cosmid K05D4, complete sequence. Caenorhabditis elegans Gaenorhabditis elegans cosmid K05D4, complete sequence. Caenorhabditis elegans Corynebacteriumn glutamicumn Ipd gene, complete 00S. Corynebacteriumn glutamicumn Drosophila melanogaster chromosome 31J69C1 clone RPCI98-1 1 N6, Drosophila melanogaster SEQUENCING IN PROGRESS 70 unordered pieces.

Drosophila metanogaster chromosome 3L-169CII clone RPC198-1 1 N6. Drosophila melanogaster -SEQUENCING IN PROGRESS 70 unordered pieces.

Eschericila coi GaIF (gaIF) gene, partial cds: 0-antigen repeat unit transporter Escherichia coli Wzx (wzx), WbnA (wbnA), 0-antigen polymerase Wzy (wzy), WbnB (wbnB), WbnG (wbnG), WbnD (wbnD), WbnE (wbnE), UDP-Glc-4-epimerase GalE (gatE). 6-phosphogluconate dehydrogenase Gnd (gnd), UDP-Glc-6dehydrogenase Ugd (ugd), and WbnF (wbnF) genes, complete cds; and chain length determinant Wzz (wzz) gene, partial cds.

Escherlchia coll hypothetical uridine-5'-dlphosphoglucose dehydrogenase (ugd) Escherlchla coti and 0-chain length regulator (wzz) genes, complete cds.

Ecoli genomic DNA, Kohara clone #351(45.1-45.5 min.). Escherichia col 38,722 35,448 35,694 100.000 37,178 37,178 59.719 23-OCT-1 998 23-Nov-98 23-Nov-98 1-Feb-99 1 6-OCT-1 999 16-OCT-i 1999 29-OCT-1 999 4759 U78086 20226 090841 144368 AC004103 215529 AC007383 215529 AC007383 13889 AE000487 53662 AL021841 36411 U00022 215767 AC009245 215767 AC009245 215767 AC009245 225851 AC007186 202291 AC007147 59,735 5-Nov-97 Homo sapiens Xp22 BAG GS-619J3 (Genome Systems Human BAG library) complete sequence.

Homo sapiens clone NHO31 OK1 5. SEQUENCING IN PROGRESS 4 unordered pieces.

Homo sapiens clone NHO31 OK15. SEQUENCING IN PROGRESS ,4 unordered pieces.

Escherichia coi K-1 2 MG1 655 section 377 of 400 of the complete genome.

Mycobacterium tuberculosis H37Rv complete genome; segment 143/162.

Mycobacterium leprae cosmid L308.

Homo sapiens chromosome 7~ 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 03.0.6 map 32A-32A strain y; cn bw sp. SEQUENCING IN PROGRESS-, 91 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR19N18 (0572) RPCI-98 19.N.18 map 32A-32A strain y; cn bw sp, -SEQUENCING IN PROGRESS 22 unordered pieces.

Homo sapiens Homo sapiens Homo sapiens Escherichia coli Mycobacterium tuberculosis Mycobacterium Ieprae Homo sapiens Homo sapiens Homo sapiens Drosophila melanogasler Drosophila melanogaster 37,904 37,340 36,385 36,385 39,494 46,252 46,368 36,016 36,016 39,618 35,366 35,366 21-MAR-1997 18-Apr-98 25-Sep-99 25-Sep-99 12-Nov-98 23-Jun-99 01-MAR-1994 2-Nov-99 2-Nov-99 2-Nov-99 07-DEC-I1999 07-DEC-i1999 2007203042 29 Jun 2007 Table 4 (continued) GBHTG3:AC010207 207890 AC010207 Hoamo sapiens clone RPCI1 1-375120. SEQUENC)NG IN PROGRESS-, 25 Homo sapiens unordered pieces.

1107 rxa01365 1497 rxa01369 1305 rxa01377 1209 rxa01392 1200 GBBA2:AF109682 GBHTG2:A0006759 GBHTG2:AC006759 GBBA1:MTY2OB11 GBBA1:XANXANAB GBGSSIO:AQ194038 GBBA1:MTY2OBi1 GBGSS3:810037 GB-GSS3:809549 GBBA1:MTCY7I GBHTG5:AC007547 GB-HTG5:AC007547 GB-BA2:AF072709 990 AF109682 Aquaspirillum arcticum malate dehydrogenase (MDH) gene, complete cds. Aquaspiriltumn arcticum 103725 AC006759 Caenorhabditis elegans clone Y400i12, 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. Mycobacteriumn tuberculosis 3410 M83231 Xanthomonas campestris phosphoglucomulase and phosphomannomnutase Xanthomonas campestris (xanA) and phosphomannose isomerase and GDP-mannose pyrophosphorylase (xanB) genes, compltee cds.

697 AQ194038 RPCII11-47D24.TJ RPCI-1 1 H-omo sapiens genomic clone RPCI-1 1-47D324, Homo sapiens genomic survey sequence.

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

1097 B09549 T21A19-T7.1 TAMU Arabldopsis thaliana genomic clone T21A1 9. genomic Arabidopsis thatiana survey sequence.

42729 Z92771 Mycobacterium tuberculosis H37Rv complete genome; segment 1411162. Mycobacterium tuberculosis 262181 AC007547 Homo sapiens clone RP1 1-252018. WORKING DRAFT SEQUENCE, 121 Homo sapiens unordered pieces.

262181 AC007547 Homo sapiens clone RP1 1-252018, WORKING DRAFT SEQUENCE, 121 Homo sapiens unordered pieces.

8366 AF072709 Streptomyces fividans amplilable 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. Corynebacterium glutamtcum 185952 AC005906 Homo sapiens 12p1 3.3 BAC RPCI 1 1-429A20 (Roswell Park Cancer Homo sapiens institute Human BAC Library) complete sequence.

3657 X89084 C.glutamicum pta gene and ackA gene. Corynebacteriumn glutamicum 14839 D90861 Ecoli genomic DNA, Kohara clone #405(52.0-52.3 min.). Escherichia coi 1200 E02087 DNA encoding acetate kinase protein form Escherlchia coli. Escherichia coli 280 U60627 Heticobacter pylon feoB-like DNA sequence, genomic survey sequence. Helicobacler pyloni 349 A1701691 we8lcO4.xl SoaresNFLTGBC_51 Homo sapiens cDNA clone H-omo sapiens IMAGE:2347494 3' similar to gb:L1 9686jnall MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);, mRNA sequence.

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 16-Sep-99 19-OCT.1999 25-Feb-99 25-Feb-99 17-Ju v-98 26-Apr-93 20-Apr-99 17-Jun-98 14-MAY-i 997 14-MAY-i 997 0) K3' 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 GBBAl :CGLYSEG GB-PR4:AC005906 rxa01436 1314 GB-BA1:CGPTAACKA GBBA:D90861 GBPAT.E02087 rxa01468 948 GB-GSS1:HPU60627 GB-EST3l :A1701691 2007203042 29 Jun 2007 Table 4 (continued) GBEST15:AA480256 389 A.A480256 ne3ltO4.sl NCICGAPCo3 Homo sapiens cDNA clone IMAGE:898975 3' rxa01478 1959 rxa01482 1998 GB-BA1 :SC151 GB-BAI:SC E36 GB-BAI :CGU43 535 GB-BA1 :SC6G4 GBBA1:U00020 GBBA1:MTCY77 40745 112581 2531 41055 36947 22255 AL109848 AL049763 U43535 AL03 1317 UJ00020 Z95389 similar to gb:L19686jrna1 MACROPHAGE MIGRATION INHIBITORY FACTOR (HUMAN);. mRNA sequence.

Streptomyces coelicolor cosmid 151.

Streptomycos coelicolor cosmld E36.

Corynebacterium glutamicum muttidrug resistance protein (cmr) gene, complete cds.

Streptomyces coelicolor cosmid 6G4.

Mycobacterium Ieprae cosmid B229.

Mycobacterium tuberculosis H37Rv complete genome; segment 146/162.

Homo sapiens Streptomyces coelicolor A3(2) Streptomyces coelicolor Corynebacterlum glutamicum Streptomyces coellcolor Mycobacterium Ieprae Mycobacterium tuberculosis 39,574 54,141 38.126 41,852 62,149 38,303 38,179 14-Aug-97 16-Aug-99 05-MAY.1999 9-Apr.97 20-Aug.98 01-MAR-1994 18-Jun-98 rxa0l 534 rxa01535 1530 rxa0l550 1635 GB-BA1:MLCB1222 GBBA1:MTVO17 GBBA1:PAU72494 GBBAI:D90907 GB_1N2:AF073177 GB_1N2:AF073179 34714 67200 4368 132419 9534 3159 ALO-49491 AL021 897 U72494 090907 AF0731 77 AF0731 79 Mycobacterium leprae cosmid B1222. Mycobacterium leprae Mycobacterium tuberculosis H37Rv complete genome: segment 48/1 62. Mycobacterium tuberculosis Pseudomonas aeruginosa fumarase (fumC) and Mn superoxide dismutase Pseudomonas aeruginosa (sodA) genes, complete cds.

Synectiocystis 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

W

23-OCT-1996 7-Feb-99 1-Jul-99 27-Apr-99 rxa01 562 rxa01569 1482 GBBAI:D78182 GBBA2:AF0791 39 GBBA2:AF087022 rxa0i570 978 GB-BA1:MTCY63 GB-BA2:AF09751 9 7836 4342 1470 38900 13781182 AF079 139 AF087022 Z96800 Streptococcus mutans DNA for dTDP-rhamnose synthesis pathway, complete cds.

Streptomyces venezuelae pikCD operon. complete sequence.

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

Mycobacterium tuberculosis H37Rv complete genome; segment 16/1 62.

Streptococcus mutans Streptomyces venezuelae Streplomyces venezuelae Mycobacterium tuberculosis Klebsiella pneumoniae 44,050 38,587 38.62 1 59,035 59.714 5-Feb-99 28-OCT-i 998 15-OCT-i1998 17-Jun-98 4-Nov-98 4594 AF097519 Klebsiella pneumonlae dTDP-D-glucose 4.6 dehydratase (QmIB), glucose-i1phosphate thymidytyl transferase (rmLA), dTDP-4-keto-L-rhamnose reductase (rmlD), dTDP-4-keto-6-deoxy-D-gluc-ose 3,5-epimerase (rmIC). and rhamnosyl Iransferase (wbbL) genes, complete cds.

2007203042 29 Jun 2007 58,3a4 30-Jul-96 GBBA2:NGOCPSPS m0a1571 723 GBBA1:AB01 1413 GBBA1:ABO1 1413 rxa01572 615 GB BA1:ABO11413 GB-BA1 :ABO 11413 8905 L09189 Table 4 (continued) Neisseria meningitidis dTDP-D-glucose 4.6-ehydratase (rlbB), glucose-i Neisseria meningitidis phosphate thymidyl transferase (rfbA) and rfbC genes, complete ccds and UPDglucose-4-epimerase (galE) pseudogene.

Streptomyces griseus genes for 0rf2. 04f3, 04f4, Orf5, AfsA, Orf8, partial and Streptomyces gniseus complete cds.

Streptomyces griseus genes for Orf2, 04 3, Or-f4, 04f5, AfsA, 04f8, partial and Streptomyces griseus complete cds.

12070 12070 AB01I1413 ABOI 1413 rxa01606 2799 GBVI:CFU72240 1207 0 A BO11413 Streptomyces griseus genes for 04f2. 043, 04I4,04r5, AfsA. 048. partial and Streptomyces griseus complete cds.

12070 AB0l 1413 Streptomyces griseus genes for 04f2. 043, 04f4, 04f5, AfsA. 04f8, partial and Streptomyces griseus complete cds.

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

408 AQ21 3248 HS_3249_BiA02_MR CIT Approved Human Genomic Sperm Library D Homo Homo sapiens sapiens genomic clone Plale=3249 001=3 Row=B. genomic survey sequence.

285 A0070145 HS_3027_81_H02_MR CIT Approved Human Genomic Sperm Library 0 Homo Homo sapiens sapiens genomic clone Plate=3027 001=3 Row=P. genomic survey sequence.

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 GBGSS1O:AQ213248 GBGSS8:A0070145 34.559 18-Sep-98 40,351 5-Aug.98 rxa01626 468 GBPR4:AF152510 GBPR4AF152323 GB PR4:AF 152509 rxa01632 1128 GBHTG4:AC006590 GBHTG4:AC006590 2490 4605 2712 127171 AF152510 AF152323 AF152509 AC006590 127171 AC006590 Homo sapiens prolocadherin gamma A3 short form protein (PCDH-gamma-A3) variable region sequence, complete cds.

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

Homo sapiens PCDH-gamma-A3 gene, aberrantly spliced, mRNA sequence.

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

Drosophila melanogaster chromosome 2 clone BACR13NO2 (ID543) RPCI-98 13.N.2 map 36E-36E strain y: cn bw sp. SEQUENCING IN PROGRESS-, 101 unordered pieces.

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

Bacillus subtilis complete genonie (section 9 of 21): from 1598421 to 1807200.

Bacillus subtilis complete genonie (section 9 of 21): from 1598421 to 1807200.

Drosophila melanogaster chromosome 2 clone BACR48110 (0D505) RPCI-98 48.1.10 map 49E6-49F8 strain y: on bw sp, SEQUENCING IN PROGRESS unordered pieces.

Homo sapiens Homo sapiens Homo sapiens Drosophila melanogasler Drosophila melanogaster Homo sapiens Bacillus subtilis Bacillus subtilis Drosophila melanogaster 34,298 34,298 34.298 33,812 33,812 36.111 36,591 34,941 37,037 14-Jul-99 22-Jul-99 14-Jul-99 19-OCT-I1999 19-OCT-i1999 26-Jun-98 26-Nov-97 26-Nov-97 2-Aug-99 rxa01633 1206 GBG558:B99182 GBBA1:BSUBOOO9 GBBAI:BSUBOOO9 GB-ITG2:AC006247 415 208780 208780 174368 B99182 Z99 112 Z991 12 AC006247 2007203042 29 Jun 2007 rxaOI695 1623 GBBAI:CGA224946 GBBA1:MTCY24A1 GB_1N1:DMU15974 nxa01702 1155 GBBA1:CGFDA GB-BA1:MTY13E10 GBBA1:MLCB4 rxa01743 901 GB_1N2:CELC27H5 GBEST24:A1167112 GBGSS9:AQ102635 rxa01744 1662 GBBA1:MTCYOIB2 GBGSS1:AF009226 GBBA1:SCD78 rxa01745 836 GBBA1:MTCY19O GBBAI:MLCB22 GB-BA2:AEOO1 75 nca01758 1140 GBPR3:HS57G9 GBPL2:YSCH9666 GBPL2:YSCH9986 rxa01814 1785 GBBA1:ABCCELB GBBA1:MTCY22D7 GBBA1:MTCY22D7 rxaO185l 1809 GB..GSS9:A0142579 GB_1N2:AC005889 GBGSSI:AGOO88I4 2408 20270 2994 3371 35019 36310 35840 579 3.47 35938 665 36224 34150 40281 115067 113872 39057 41664 2058 31859 31859 529 108924 637 AJ224946 Z95207 U 15974 X17313 Z95324 AL023514 U14635 A1167112 AQ102635 Z95554 AF009226 AL034355 Z70283 Z98741 AECO00175 Z95116 U 10397 U00027 L24077 Z83866 Z83866 AQ142579 AC005889 AG00881 4 Table 4 (continued) Corynebacterium glutamicum DNA for L-Melate:qulnone oxidoreductase.

Mycobacterium tuberculosis H37Rv complete genome; segment 124/162.

Drosophila melanogaster kinesin-like protein (klpG8d) mRNA, complete cds.

Corynebacterlumn glutamicum fda gene for fructose-bisphosphate aldolase (EC 4.1.2. 13).

Mycobacterlumn tuberculosis H37Rv complete genome segment 18/162.

Mycobacterium leprae cosmid B4.

Caenorhabditis elegans cosmid C271-5.

xylem.est.878 Poplar xylem Lambda ZAPII library Populus balsamifera subsp.

trichocarpa cDNA mRNA sequence.

HS_3048_81_F08_MF CIT Approved Human Genomlc Sperm Library D Homo sapiens genomic clone Plate=3048 001=1 5 Row=L, genomic survey sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 72/1 62.

Mycobacteriumn tuberculosis cytochrome D oxidase subunit I (appO) gene.

partial sequence, genomic survey sequence.

Streptomyces coelicolor cosmid D78.

Mycobacterium tuberculosis H37Rv complete genome; segment 98/1 62.

Mycobacteriumn leprae cosmid 822.

Escherichia coli K-1 2 MG1 655 section 65 of 400 of the complete genome.

Human DNA sequence from BAG 57G9 on chromosome 22q12.1 Contains ESTs, CA repeat. GSS.

Saccharomyces cerevisiae chromosome VIII cosmid 9666.

Saccharomyces cerevisiae chromosome VIII cosmid 9966.

Acetobacter xylinumn phosphoglucomnulase (ceIB) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 133/1 62.

Mycobacteriumn tuberculosis H37Rv complete genome: segment 133/1 62.

Corynebacterium glutamicumn Mycobacteriumn tuberculosis Drosophila melanogaster Corynebactenumn glutamicum' Mycobacteriumn tuberculosis Mycobacterlumn Ieprae Caenorhabditis elegans Populus balsamifera subsp. trlchocarpa Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis Streptomyces coelicolor Mycobacteriumn tuberculosis Mycobacterium Ieprae Escherichia coi Homo sapiens 100,000 38,626 36.783 99,913 38,786 38,238 35,334 39,222 40,653 36,650 63.438 53.088 62,081 61.364 52,323 39,209 11 -Aug-98 17-Jun-98 18-Jul-95 12-Sep-93 17-Jun-98 27-Aug-99 13-Jul-95 03-DEC-i1998 27-Aug-98 31-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 Saccharomyces cerevislae 40,021 Sacclaromyces cerevisiae 34,375 Acetobacter xylinus 62,173 Mycobacteriumn 39,749 tuberculosis Mycobacterlumn 40.034 tuberculosis HS_2222_81_H03_MR CIT Approved Human Genomic Sperm Library D Homo H-omo sapiens sapiens genomic clone Plate=2222 Col=5 Row=P, genomlc survey sequence.

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

Homo sapiens genomic DNA, 21Iq region, clone: 13 7878868, genomic survey Homo sapiens sequence.

38,068 24-Sep-98 36.557 35,316 30-OCT- 1998 7-Feb-99 2007203042 29 Jun 2007 rxa0l859 1050 GB-BA2:AF 183408 GB-HTG:AC008031 GBBA2:AF183408 rxa01865 438 GBBA1:SERFDXA GB-BA1 :MTVOO5 63626 AF183408 158889 AC008031 63626 AF183408 3869 M61119 Table 4 (continued) Microcystis aewuginosa DNA polymerase IlI beta subunit (dnaN) gene, partial cds; microcystin synthetase gene cluster, complete sequence; Umal (uma I), Uma2 (uma2), Uma3 (uma3), Uma4 (uma4l), and UmaS (uma5) genes, complete cds; and Uma6 (uma6) gene, partial cds.

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

Microcystis aeruginosa DNA polymerase III beta subunit (dnaN) gene, partial cds: micbocystin synthetase gene cluster, complete sequence; Umal (uma 1), Uma2 (uma2), Uma3 (uma3l), Uma4 (uma4l), and Uma5 (uma5) genes.

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

Saccharopoyspora erythraea ferredoxin (fdxA) gene, complete cds.

37840 AL010186 Mycobacterlum tuberculosis H37RY complete genome: segment 51/162.

Microcystis aeruginosa Trypanosoma brucei Miciocyslis aeruginosa Saccharopolyspora erythraea Mycobacteriumn tuberculosis Mycobacterium tuberculosis Homo sapiens Homo sapiens Homo sapiens Mycobacteriumn tuberculosis Streptomyces coelicolor 36.364 35.334 15-Nov-99 36,529 59,862 03-OCT. 1999 13-MAR-i1996 03-OCT. 1999 61,949 17-Jun-98 59,908 10-DEC-1996 GBBA1:M5GY348 40056 AD000020 Mycobacterium tuberculosis sequence from clone y348.

nma01882 1113 GBPR1:HUMADRA2C GBPR4:H5U72648 GB-GSS3:B42200 1491 J03853 4850 U72648 387 B42200 Human kidney alpha-2-adrenergic receptor mRNA. complete cds.

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

HS-1055-B31-A0-MR.abi CIT Human Genomic Sperm Library C Homo sapiens genomic clone Plate'=CT 777 Col=5 Row--B, genomic survey sequence.

36.899 36,899 34,805 37,892 40,413 27-Apr-93 23-Nov-98 18-OCT-1997 1 7-Jun-98 29-MAR-1 999 rxa01884 1913 GB-BA1 :MTCY48 35377 Z74020 Mycobacterium tuberculosis H37Rv complete genome; segment 69/162.

GBBA1:5C0001206 9184 AJO01206 Streptomyces coelicolor A3(2), glycogen metabolism cluster 11.

GBBA1:D90908 rxa01886 897 GBGSS9:AO1 16291 122349 090908 572 AQ1 16291 rxa01887 1134 GBBA2:AE001721 17832 AE001721 GBE5T16:AA567090 596 AA567090 GBl-HTG6:AC008147 303147 AC008147 GB-HTG6:AC008147 303147 AC008147 GSBBA2:ALW243431 26953 AJ243431 GBI-HTG2:ACOO8I 97 125235 AC0081 97 Synechocystis sp. PCC6803 complete genome, 10/27, 1188886-1311234. Synechocystis sp.

RPCII1 49P6.TK.1 RPCI-1 1 Homo sapiens genomic clone RPCI-1 1-49P6, Homo sapiens genomic survey sequence.

Therrnotoga maritima section 33 of 136 of the complete genome. Thermotoga mariti GM01O44.5prime GM Drosophila melanogaster ovary BlueScript Drosophila Drosophila melano melanogastercDNA clone GM01044 Sprime, mRNA sequence.

Homo sapiens clone RP3-405J110. SEQUENCING IN PROGRESS 102 Homo sapiens ma 39.306 gaster 42,807 2-Jun-99 28-Nov-98 47,792 7-Feb-99 43,231 20-Apr-99 unordered pieces.

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

Acinetobacter Iwoffii wzc, wzb, wza, weeA, weeB, wceC, wzx. wzy, weeD, Acinetobacter Iwoffii weeE, weeF, weeG, weeH, weel, weeJ, weeK. galU, ugd, pgi, galE, pgm (partial) and mip (partial) genes (emulsan biosynthetic gene cluster), strain RAG-i.

Drosophila mnelanogaster chromosome 3 clone BACRO2112 (0753) RPCI-98 Drosophila melanogaster 021L.12 map 9413-94C strain y; cn bw sp, SEQUENCING IN PROGRESS-, 113 unordered pieces.

36.417 03-DEC-i 999' 37,667 03-DEC-1999 39,640 01-OCT-1999 rxa01888 658 32.969 2-Aug-99 2007203042 29 Jun 2007 GBHTG2:AC008197 125235 AC008197 Table 4 (continued) Drosophila melanogaster chromosome 3 clone BACRO212 (D753) RPCI-98 Drosophila melanogaster 32,969 021L.12 map 94B-94C strain y; on bw sp, SEQUENCING IN PROGRESS 2-Aug-99 GBEST36:A1881527 rxaOI89l 887 GBVI:H1V232971 GB PL1 AFOI-SE GBPR3:AF064858 rxa01895 1051 GBBA1:CGL238250 GB.BA2:AF038423 GBBA1:MTCY359 rxa~l9DI 1383 GBBA1:MSGB3800S OB-BAI :SCE63 GBPR3:AF093117 rxa01927 1503 GBBA1:CGPAN GBBA1:ASXYLA GB-HTG3:AC009500 rxa01952 1836 GB_8A2:AE000739 GBEST28:A1519629 GB-EST21 :AA949396 rxa01989 630 GBBA1:BSPGIA GBBAI:.BSUBOO17 GBBA2:AF132127 rxa02026 720 GB-BA1:SXSCRBA GBBA1:BSUBOO2O GBBA1:BSGENR rxa 02028 526 GB-BAI :MTC1237 598 621 6158 193387 1593 1376 36021 37114 37200 147216 2164 1905 176060 13335 612 767 1822 217420 8452 3161 212150 97015 27030 A188 1527 AJ232971 Y09542 AF064858 AJ238250 AF038423 Z83859 L01095 AL035640 AF093 117 X96580 X59466 AC009500 AE000739 A1519629 A949396 X1 6639 Z99120 AF132127 X67744 Z99123 X73 124 Z94752 **,113 unordered pieces.

606070C09.y 1 606 Ear tissue cDNA library from Schmidt lab Zea mays cDNA,Zea mays mRNA sequence.

Human immunodeficlency virus type 1 subtype C net gene, patient MP83. Human immunodeficiency virus type 1 Afumigatus chsE gene. Aspergillus fumigatus Homo sapiens chromosome 21q22.3 BAC 28F9, complete sequence. Homo sapiens Corynebacterium glutamicum ndh gene. Corynebacterum glulamicum Mycobacterium smegmatis NADH dlehydrogenase (ndh) gene, complete eds. Mycobacterlum smegmatis, Mycobacterium tuberculosis H37Rv complete genome; segment 84/1 62. Mycobacterium tuberculosis M. leprae genomic DNA sequence, cosmid B38 bfr gene, complete cds. Mycobacterium leprae Streptomyces coelicolor cosmid E63. Streptomyces coelicolor Homo sapiens chromosome 7qtelo BAC E3, complete sequence. Homo sapiens C.glutamicum panB, panC xylB genes. Corynebacterium glutamicum Arthrobacter Sp. N.R.R.L. B3728 xyLA gene for D-xylose(D-glucose) isomerase. Arthrobacter sp.

Homo sapiens clone NHO511IA2O,** SEQUENCING IN PROGRESS 6 Homo sapiens unordered pieces.

Aquifex aeolicus section 71 of 109 of the complete genome. Aquifex aeolicus LD39282.Sprime LD Drosophila melanogaster embryo pOT2 Drosophila Drosophila melanogaster melanogaster cDNA clone LD39282 5prime, mRNA sequence.

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

Bacillus stearothennophilus pgiA gene for phosphoglucolsomerase isoenzyme Bacillus A (EC stearothermophilus Bacillus subtills complete genome (section 17 of 21): from 3197001 to Bacillus subtilis 3414420.

Streptococcus mutans sorbitol phosphoenolpyruvate:sugar phosphotransferase Streptococcus mutans operon. complete sequence and unknown gene.

S.xylosus scrB and scrR genes. Staphylococcus xylosus Bacillus subtilis complete genome (section 20 of 21): from 37984011to Bacillus subtilis 4010550.

B.subtilis genomic region (325 to 333). Bacillus subtilis Mycobacterium tuberculosis H37Rv complete genome; segment 46/162. Mycobacterium tuberculosis 43.6 17 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-i1999 1-Apr.97 2-Jun-98 24-Apr-99 05-MAY-i1998 17-Jun-96 6-Sep-94 17-MAR-1999 02-OCT-1998 11-MAY-i1999 04-MAY- 1992 24-Aug-99 25-MAR-1 998 16-MAR-1999 25-Nov-98 20-Apr-95 26-Nov-97 28-Sep-99 28-Nov-96 26-Nov-97 2-Nov-93 17-Jun-98 2007203042 29 Jun 2007 GBPL2:SCE9537 GB-GSS13:A0501 177 rxa02054 1140 GBBA1:MLCB1222 GBBA1:MTY13EI2 GB-BA1 :MTU43SAO rxa02O56 2891 GB-PAT:E14601 GBBAI:D84102 GBBA1:M1V006 rxaO2O61 1617 GBHTG7:AC005883 GBPL2:ATACOO3O33 GBPL2:ATAC002334 rxa02O63 1350 GBBA1:SCGLGC GB-GSS4:AQ687350 GBEST38:AW028530 rxaO21OO 23,48 GB-BAI:MSGY151 GB.BA1 :MTCY1 30 GBBA1:SCOOO12OS rxa02122 822 GBBA1:D90858 GBEST37:A1948595 GBHTG3:AC010387 66030 767 34714 43401 3453 4394 4394 22440 211682 84254 75050 1518 786 444 37036 32514 9589 13548 469 220665 U 18778 AQ501177 AL049491 Z95390 U43540 E14601 084102 AL021 006 AC005883 AC003033 AC002334 X89733 A0687350 AW028530 AD00001 8 Z73902 AJO01 205 D90858 A1948595 AC01 0387 178813 L78814 AF093099 Z70283 Table 4 (continued) Saccharomyces cerevislae chromosome V cosmlds 9537, 9581. 9495, 9867, Saccharomyces cerevisiae 36,100 and lambda dlone 5898.

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

Mycobacterium leprae cosmid 81222. Mycobactefium leprae 1-Aug-97 29-Apr-99 Mycobacterium tuberculosis H37Rv complete genome: segment 147/162. Mycobactenumn tuberculosis Mycobacterium tuberculosis rfbA, rhamnose biosynthesis protein (rfbA), and Mycobacterium rmlC genes, complete cds. tuberculosis Brevibacterium lactofermentum gene for alpha-ketoglutaric acid Corynebacterium dehydrogenase. g lutamicum Corynebacterlum glutamicum DNA for 2-oxoglutarate dehydrogenase, complete Corynebacterium cds. gtutamicum Mycobacterium tuberculosis H37Rv complete genome; segment 54/162. Mycobacterium tuberculosis Homo sapiens chromosome 17 clone RPI 1-958E11I map 17, Homo sapiens SEQUENCING IN PROGRESS 2 ordered pieces.

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

Arabldopsis thaliana chromosome 11 BAG F25118 genomic sequence, complete Arabidopsis thaliana sequence.

Scoelicolor DNA for gtgC gene. Streptomyces coeticolor nbxbOO74H-1 1r CUGI Rice BAG Library Oryza saliva genornic clone Oryza sativa nbxbOO74H1 1r, genomic survey sequence.

wv27f IO.xl NCL-CGAP-id1 1 Homo sapiens cDNA clone IMAGE:2530795 3' Homo sapiens similar to WP:TO3G1 1.6 CE04874 mRNA sequence.

Mycobacterium tuberculosis sequence from clone yl51. Mycobacterium tuberculosis Mycobacterium tuberculosis H-37Rv complete genome; segment 59/162. Mycobacterium tuberculosis Streptomyces coelicolor A3(2) glycogen metabolism clusterl. Streptomyces coelicolor Ecoli genomic DNA, Kohara clone #401(51.3-51.6 min.). Escherichla coi wqO7dl2.xl NCI-CGAPKid 12 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. Mycobacterium leprae Mycobacterium leprae cosmid 81554 DNA sequence. Mycobacterium leprae Mus musculus transcription factor TBLYM (Tblym) mRNA, complete cds. Mus muscuius Mycobacterium tuberculosis H37Rv complete genome: segment 98/1 62. Mycobacterium tuberculosis 61.896 59.964 59,659 98,928 98,928 39.265 37.453 37,711 37,711 56.972 40,696 36,795 40,156 55,218 38,475 38,586 37,259 38,868 51,399 51.399 36,683 57,292 27-Aug-99 1 7-Jun-98 14-Aug-97 28-Jul-99 6-Feb-99 18-Jun-98 08-DEC-1999 19-DEC-1997 04-MAR-i1998 12-Jul-99 11-Jul-99 27-OCT.1999 10-DEC-1996 '17-Jun-98 29-MAR-i 999 29-MAY-i1997 6-Sep-99 1 5-Sep-99 15-Jun-96 15-Jun-96 01-OCT-i1999 17-Jun-98 rxaO214O 1200 rxa02142 774 GB-BA1:MSGB1551CS 36548 GB BAI:MSGB1 S54CS 36548 GBRO:AF093099 2482 GB-BAI:MTCY190 34150 2007203042 29 Jun 2007 Table 4 (continued) Streptomyces coelicolor cosmid 6G1 GB_BA1:SC6G10 36734 AL049497 GBBA1:AB016787 5550 ra02143 1011 GBBA1:MTCY19O 34150 GBBA1:MSGB1551CS 36548 GBBA1:MSGBI554CS 36548 nxa02144 1347 GBBA1:MTCYI90 34150 GB HTG3:AC01 15000 0300851 GB-HTG3:ACO1 15000 300851 nxa02147 1140 GBEST28:A1492095 GBEST1O:AA157467 GBEST1O:AA157467 rxa02149 1092 GB-PR3:HSBK277P6 GB_BA2:EMB065R075 GBEST34:A1789323 r)(a02175 1416 GBBAi:CGGLTG GBBA1:MTCY3I GBBAI-MLCB57 rxa02196 816 GB_RO:RATDAPRP GBGSS8:AQ012162

GBRO:RATDAPRP

rxa02209 1694 GBBAI:AB025424 GBBA2:AF002133 485 376 376 61698 360 574 3013 37630 38029 2819 763 2819 2995 15437 ABO016787 Z70283 L78813 L78814 Z70283 ACOI 1500 ACO 1500 A1492095 AA1 57467 AA1 57467 AL1 17347 AFI 16423 A1789323 X66 112 273101 Z99494 M76426 AQ012 162 M76426 AB02-5424 AF002 133 Pseudomonas putida genes for cytochrome o ubiquinol oxidase A-E and 2 ORFs, complete cds.

Mycobacterium tuberculosis H-37Rv complete genome; segment 981162.

Mycobaclerium leprae cosmid 131551 DNA sequence.

Mycobacterium leprae cosmid 131554 DNA sequence.

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

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

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

tgO7a0l x1 NCL.CGAP_CLL1I Homo sapiens cONA clone IMAGE:2108040 3T, mRNA sequence.

zo50e0l.rl Stratagene endothelial cell 937223 Homo sapiens cONA clone IMAGE:590328 mRNA sequence.

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

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

complete sequence.

Rhizobium etli mutant MB045 RosR-transcriptionally regulated sequence.

uk53gOS.yl Sugano mouse kidney mkia Mus musculus cDNA clone IMAGE:1972760 5' similar to WP:K1 1H12.8 CE12160 mRNA sequence.

C.glutamicum gIl gene for citrate synthase and ORF.

Mycobacterium tuberculosis H37Rv complete genome; segment 4 1/1 62.

Mycobacterium leprae cosmid B57.

Rattus norvegicus dipeptidyt aminopeptidase -related protein (dpp6) mRNA, complete cds.

127PB037070197 Cosmid library of chromosome 11 Rhodobacter sphaeroides genomic dlone 127PB037070197, genomlc survey sequence.

Rattus norvegicus dipeptldyl aminopeptidase-retated protein (dpp6) mRNA, complete cds.

Corynebacterium glutamicum gene for aconltase, partial cds.

Mycobacterium avium strain GIR10 transcriptional regulator (may81) gene, partial cds. aconitase (acn). invasin 1 (Invi), Invasin 2 (inv2), transcriptional regulator (moxR), ketoacyl-reductase (fabG), enoyl-reductase (inhA) and ferrochelatase (mav272) genes, complete cds.

Streptomyces coelicolor Pseudomonas putida Mycobacterium tuberculosis Mycobacterium leprae Mycobacterium leprae Mycobacterium tuberculosis Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Rhizobium etli Mus musculus Corynebacterium glutamicum Mycobacterium tuberculosis Mycobacterium leprae Rattus norvegicus Rhodobacler sphaeroides Rattus norvegicus Corynebactedrn glutamicum Mycobacterium avium 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 62,49 1 38,791 40,044 37,312 99,173 40,219 5-Aug-99 17-Jun-98 15-Jun-96 1 5-Jun-96 17-Jun-98 18-Feb-00 18B-Feb-00 30-MAR-i1999 I 1-DEC-1996 11-DEC-1996 23-Nov-99 06-DEC-I1999 2-Jul-99 17-Feb-95 17-Jun-98 10-Feb-99 31-MAY- 1995 4-Jun-98 31-MAY-i1995 3-Apr-99 26-MAR-I1998 35,058 24-MAR-i1999 2007203042 29 Jun 2007 GBBA1:MTVOO7 rxa02213 874 GBBA1:AB025424 GBBA1:M1V007 GBBA2:AF002 133 rxa02245 780 GB-BA2:RCU23145 GBBA1:ECU82664 GBHTG2:AC007922 rxa02256 1125 GBBA1:CGGAPPGK GB-BA1 :SCC54 GBBAI:MTCY493 rxa02257 1338 GBBAI:CGGAPPGK GBBA1:MTCY493 GBBA2:MAU82749 rxa02258 900 GBBAl :CGGAPPGK GBBA1:CORPEPC GByPAT:A09073 rxa02259 2895 GBBA 1:CORPEPC GBPAT:A09073 GBBA1:CGPPC 32806 2995 32806 15437 AL021 114 AB025424 AL021184 AF0021 33 5960 U23145 139818 U82664 158858 AC007922 3804 30753 40790 3804 40790 2530 3804 4885 4885 4885 4885 3292 X59403 AL035591 Z95844 X59403 Z95844 U82749 X59403 M258 19 A09073 M258 19 A09073 X14234 Table 4 (continued) Mycobacleriumn tuberculosis H37Rv complete genome: segment 64J162. Mycobacteriumn tuberculosis Corynebacterium glutamicum gene for aconilase, partial cds. Corynebacterium glutamicum Mycobacteriumn tuberculosis H37Rv complete genome; segment 641162. Mycobacterium tuberculosis Mycobacterium avlum strain GIRIO ttanscriptional regulator (may81) gene. Mycobacterium aviumn 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 opeton: fructose- Rhodobacter capsulatus 1 ,6-/sedoheptulose- 1.7-bisph osph ate aldolase (cbbA) gene, partial cds. Form 1I ribulose- 1,5-bisphosph ate carboxylase/oxygenase (cbbM) gene, complete ods, and Calvin cycle operon: pentose-5-phosphate-3-epimerase (cbbE), phosphoglycolate phosphatase (cbbZ), and cbbY genes, complete cds.

Escherichia coli minutes 9 to 11 genomic sequence. Escherichia coli Homo sapiens chromosome 18 clone hRPK.178_Fj0 map 1,Homo sapiens SEQUENCING IN PROGRESS 11 unordered pieces.

C.glutamicum gap, pgk and tpi genes for glyceraldehyde-3-phosphate, Corynebacterium phosphoglycerate kinase and triosephosphate isomerase. glutamicum Streptomyces coelicolor cosmid C54. Streplomyces coelicolor Mycobacterium tuberculosis H-37Rv complete genome: segment 631162. Mycobactedrn tuberculosis C.glutamlcum gap, pgk and tpl genes for glyce raid ahyde-3-phos ph ate. Corynebaclerium phosphoglyce rate kinase and triosephosphate isomerase. glulamicum Mycobacterium tuberculosis H37Rv complete genome; segment 63/162. Mycobacteriumn tuberculosis Mycobacterium avium glyceraldehyde-3-phosphate dehydrogenase homolog Mycobacteriumn avium (gapdh) gene, complete cds; and phos phoglyce rate kinase gene, partial cds.

C.glulamicum gap. pgk and tpi genes for glyceraldehyde-3-phosphale, Corynebacterlumn phosphog lyce rate kinase and triosephosphate isomerase. glutamicum Oglutamicumn phosphoenolpyruvate carboxylase gene, complete cds. Corynebacterium glutamicuni C.glulamicum ppg gene for phosphoenol pyruvate carboxylase. Corynebacteriurn glutarnicum C.glulamioum phosphoenolpyruvate cartboxylase gene, complete cds. Corynebacterium glutamicum C.glulamicum ppg gene for phosphoenol pyruvate carboxylase. Corynebacterium glutamlcum Corynebacteriumn glutamicumn phosphoenolpyruvate carboxylase gene (EC Corynebacteriuni 4.1.1.31). glutamicum 38,253 99,096 34,937 36.885 48,701 39.119 33,118 99,289 36,951 64,196 98.873 61,273 61,772 99,667 100,000 '100,000 1100,000 100,000 99.827 17-Jun-98 3-Apr.99 17-Jun-98 26-MAR-i1998 28-OCT-I1997 11 -Jan-97 26-Jun-99 05-OCT-I1992 1 1-Jun-99 19-Jun-98 05-OCT-1 992 19-Jun-98 6-Jan-98 05-OCT-1992 15-DEC-1995 25-Aug-93 15-DEC.1995 25-Aug-93 12-Sep-93 2007203042 29 Jun 2007 rxa02288 969 GBPR3:HSDJ94E24 GBHTG3:ACO10091 GBHTG3:ACO10091 rxa02292 798 GBBA2:AF125164 GB-GSS5:A0744695 GBEST14:AA381925 rxa02322 511 GB-BAI:MTCY22G8 GB8BA1:MTCY22G8 243145 AL050317 ra02326 939 rxa02327 1083 rxa02328 1719 rxa02332 1266 rxa02333 1038 GB_BA1:CGPYC GB-BA2:AF038548 GB-BA1 :MTCY349 GB_BA1:CGPYC GBBA2:AF038548 GBBAI:MTCY349 GB-BAI :CGPYC GBBA2:AF038548 GBPL2:AF097728 GBBAI :MSGLTA GBBA2ABU85944 GBBA2:AE000175 GBBA1:MSGLTA 159526 159526 26443 827 309 22550 22550 3728 3637 43523 3728 3637 43523 3728 3637 3916 1776 1334 15067 1776 AC010091 AC010091 AF12 5164 A0744695 AA381 925 Z95585 Z95585 Y09548 AF038548 Z83018 Y09548 AF038548 Z83018 Y09548 AF038548 AF097728 X60513 U85944 AE000175 X60513 Table 4 (continued) Human DNA sequence from clone RPI-94E24 on chromosome 20q12, complete sequence.

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

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

Bacteroictes fragilis 638R polysaccharide B (PS 832) biosynthesis locus, complete sequence; and unknown genes.

HS_5505 A2 COS SP6 RPCI-1 1 Human Male BAC Library Homo sapiens genomic clone Plate=1081 001=12 Row--E, genomic survey sequence.

EST95058 Activated T-ceils I Homo sapiens cDNA 5Fend, mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.

Mycobacterium tuberculosis H37Rv complete genome; segment 49/162.

Corynebacterium glutamicum pyc gene.

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

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

Corynebacterium glutamlcum pyc gene.

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

Mycobacterium tuberculosis H37Rv complete genome; segment 131/162.

Corynebacterium glulamicum pyc gene.

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

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

M.smegmatis gItA gene for citrate synthase.

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

Escherichia coli K-12 MG1655 section 65 of 400 of the complete genome.

M.smegmatis gltA gene for citrate synthase.

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

Homo sapiens Homo sapiens Homo sapiens Bacleroides fragilis Homo sapiens Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis Corynebacterium glutamicum Corynebacterium glutamicum Mycobacterium tuberculosis Corynebacterium glutamicum Corynebacterlumn glutamicum Mycobacterium tuberculosis Corynebacterium glutamicum Corynebacterium glutamicum Aspergillus terreus 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 41,317 100,000 100,000 36.039 03-DEC-1999 11I-Sep-99 11 -Sep-99 01-DEC-1999 1 6-Jul-99 21-Apr-97 17-Jun-98 17-Jun-98 08-MAY-i1998 24-DEC-i1997 17-Jun-98 08-MAY-i1998 24-DEC-i1997 17-Jun-98 08-MAY- 1998 24-DEC-I1997 52.248 29-OCT-1998 Mycobacterium smegmatis 58,460 Antarctic bacterium 0S2- 57,154 3R Escherichla coli 38,164 Mycobacterium smegmatis 58,929 Homo sapiens 33.070 20-Sep-91 23-Sep-97 12-Nov-98 20-Sep-91 23-Nov-99 GBPR4:HUAC002299 171681 AC002299 2007203042 29 Jun 2007 GB-HTG2:AC007889 rxa02399 1467 GB-BA1:CGACEA GB-BA1:CORACEA GBPAT:113693 nca02404 2340 GB-BA1:CGACEB

GBBAI:CORACEB

GBBA1:PFFC2 rxa02414 870 GBPR4:AC007102 GBHTG3:AC01 1214 GB-HTG3:ACOI 1214 rxa02435 681 GBBA2:AF101055

GB-OM:RABPKA

GB-OM:RABPLASISM

mx02440 963 GB-EST14:AA417723 GBEST11i:AA215428 GB-BA1 :MTCY77 rxa02453 876 GB-EST14:AA426336 GBBA:STMMACC8 GB-PR3:AC004500 rxa02474 897 GB-BAI:AB009078 GBOM:BTU71200 GBEST2:F12685 rxa02480 1779 GB-BA1:MTV012 127840 AC007889 Table 4 (continued) Drosophila melanogaster chiromosome 3 clone BACR48E 12 (D695) RPCI-98 Drosophila, melanogaster 3A4.897 48.E.12 map 87A-87B strain y; cn bw sp, SEQUENCING IN PROGRESS-, 86 unordered pieces.

2427 1905 2135 3024 2725 5588 176258 183414 183414 7457 4441 4458 374 303 22255 375 1353 77538 2686 877 287 70287 X75504 128760 1 13693 X78491 L27123 Y11998 AC007102 AC011214 AC0 11214 AF101055 J03247 M64656 A417723 AA21 5428 Z95389 A426336 M55426 AC004500 ABO09078 U71200 F 12685 AL02 1287 Oglutamicum accA gene and thiX genes (partial).

Corynebacterium glutamicum isocitrate lyase (aceA) gene.

Sequence 3 from patent US 5439822.

C.glutamicum (ATCC 13032) aceB gene.

Corynebacterium glutamicumn malate synthase (aceB) gene, complete cds.

P.fluorescens FC2.1, FC2.2, FC2.3c, P02.4 and FC2.5c open reading frames.

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

Homo sapiens clone 5_-C LOW-PASS SEQUENCE SAMPLING.

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

Clostridium acetobutylicum atp operon, complete sequence.

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

Oryctolagus cuniculus phosphorylase kinase alpha subunit mRNA, complete eds.

zvOlbl 2.sl NCIOGAPGCB1 Homo sapiens cONA clone IMAGE:746207 3' similar to contains Alu repetitive element;conlains element Li repetitive element mRNA sequence.

zr95aO7.s1 NCI CGAP GCB1 Homo sapiens cDNA clone IMAGE:683412 3' similar to contains Alu repetitive element:, mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 146/162.

zv53g02.sI Soares-testisN-T H-omo sapiens cODNA clone IMAGE:757394 3', mRNA sequence.

S.fradiae aminoglycoside acetyltransfe rase (aacC8) gene, complete cds.

Homo sapiens chromosome 5, P1 clone 1076B9 (LBNL H-14), complete sequence.

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

Bos taurus acetoin reductase mRNA, complete cds.

HSC3DAO31 normalized infant brain cDNA H-omo sapiens cDNA clone c- 3da03, mRNA sequence Mycobacterium tuberculosis H37Rv complete genome: segment 132/1 62.

Corynebacterlum 100,000 glutamicum Corynebacterium 100,000 glutamicum Unknown. 99,795 Corynebacteriumn 99,914 glutamicum Corynebacteriurn 99,786 glutamicum Pseudomonas fluorescens 63,539 Homo sapiens 35,069 Homo sapiens 36,885 Homo sapiens 36,885 Clostridium acetobulylicum 39,605 Oryctolagus cuniculus 36.061 Oryctolagus cuniculus 36,000 Homo sapiens 38,770 2-Aug-99 9-Sep-94 10-Feb-95 26-Sep-95 13-Jan-95 11-Jul-97 2-Jun-99 03-OCT-I1999 03-OCT-i1999 03-MAR-1999 27-Apr-93 22-Jun-98 1 8-OCT-1 997 13-Aug-97 16-OCT-i1997 05-MvAY-1 993 30-MAR-I1998 13-Feb-99 8-Oct-97 14-Mar-95 23-Jun-99 Homo sapiens Mycobacterium tuberculosis Homo sapiens Streptomyces fradiae Homo sapiens Brevibacterlum saccharolytlcum Bos taurus Homo sapiens Mycobacterium tuberculosis 39,934 38,889 38,043 37,097 33,256 96,990 51,659 41.509 36.737 2007203042 29 Jun 2007 GB_BA1:SC6G1O GBBA1:APOOO6O rxa02485 rxa02492 8-40 GBBA1:STMPGM GB-BA1 :MTCY2OG9 GB_BA1:U00018 rxa02528 1098 GB_PR2:HS161NIO GB-HTG2:AC008235 GBHTG2:AC008235 36734 347800 921 37218 4299 1 56075 136017 AL049497 AP000060 M83661 Z77 162 U00018 AL008707 AC008235 Table 4 (continued) Streptomyces coelicolor cosmid 6G 1.

Aeropyrum pernix genomic DNA, section 3Y7.

Streptomyces coelicolor Aeropyrum pernix 136017 AC008235 rxa02539 1641 GBBA2:RSU17129 GBBAI:MTVO38 GBBA2:AF068264 17425 16094 3152 rxa02551 483 GBBA1:BACHYPTP 17057 GBBA1:BACHUThVAPAQ8954 GBBA1 :BSGBGLUC 4290 rxa02556 1281 GBHTG3:AC008128 335761 GBHTG3:AC008128 335761 GB-PL2:AC005292 99053 rxa02560 990 GB_INI:CEFO7AI1 35692 GBEST32:A1731605 566 GB_INI:CEF07AI1 35692 U1 7129 AL021 933 AF068264 029985 D31856 Z3-4526 AC008 128 AC008 128 AC005292 Z6651 1 A1731 605 Z66511 Streplomyces coelicolor phosphoglycerate mutase (PGM) gene, complete cds.

Mycobacterium tuberculosis H37Rv complete genome; segment 25/162.

Mycobacterium leprae cosmid B2168.

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

EST.

Drosophila melanogaster chromosome 3 clone BACRI5B19 (13995) RPCI-98 15.13.19 map 94F-95A strain y; cn bwsp, -*~SEQUENCING IN PROGRESS **.125 unordered pieces.

Drosophila melanogaster chromosome 3 clone BACR151B19 (0995) RPCI-98 15.B.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.

Mycobacleriumn tuberculosis H37Rv complete genome; segment 24/162.

Pseudamnonas aeruginosa quinoprotein ethanol dehydrogenase (exaA)gene.

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

Bacillus subtlils wapA and art genes for wall-associated protein and hypothetical proteins.

Bacillus subtilis genome containing the hut and wapA loci.

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

Homo sapiens, SEQUENCING IN PROGRESS 106 unordered pieces.

Homo sapiens, SEQUENCING IN PROGRESS 106 unordered pieces.

Genomic sequence for Arabidopsis thaliana BAC F26F24, complete sequence.

Caenorhabdftis elegans cosmid F07AI 1, complete sequence.

BNLGHi1O 201 Six-day Cotton fiber Gossypiumn hirsutum cONA 5' similar to (AC004684) hypothetical protein [Arabidopsis thaliana), mRNA sequence.

Caenorhabdltls elegans cosmid FOTAl 1, complete sequence.

Streptomyces coelicolor Mycobacterium tuberculosis Mycobacterium Ieprae Homo sapiens Drosophila melanogaster 35,511 48,014 65,672 61,436 37,893 37,051 36,822 Drosophila melanogaster 35,822 Rhodococcus erythropolis Mycobacteriurn tuberculosis Pseudomonas aeruginosa Bacillus sublilis Bacillus subtilis Bacillus subtilis Homo sapiens Homo sapiens Arabidopsis thaliana Caenothabditis elegans Gossypiumn hirsutumn Caenorhabdltls elegans 66,117 65,174 65,448 53,602 53,602 53,602 34,022 34.022 33,858 36,420 38,095 33.707 24-MAR-i1999 22-Jun-99 26-Apr-93 17-Jun-98 01-MAR-1994 23-Nov-99 2-Aug-99 2-Aug-99 16-Jul-99 17-Jun-98 18-MAR-1 999 7-Feb-99 7-Feb-99 3-Jul-95 22-Aug-99 22-Aug-99 16-Apr-99 2-Sep-99 11-Jun-99 2-Sep-99 2007203042 29 Jun 2007 rxa02572 668 GBBA1:MTCY63 GB-BA1 :MTCY63 GB-HTG1 :HS24H01 rxa02596 1326 GB-BA1:M7VO26 GBBA2:AF026540 GB_8A2:MTU96128 rxaO2611 1775 GBBA1:MTCYI3O GBBA1:MSGYI51 GBBA1:U00014 rxa02612 2316 GB-BA1:MTCY130 GBBA1:MSGY151 GBBA1:STMGLGEN rxa02621 942 GBBA1:CGL133719 GBINI:CEMIO6 GBEST29:A1547662 rxa02640 1650 GB-BA1:MTVO25 GBBAI :PAU49666 38900 Z96800 38900 Z96800 46989 AL121632 23740 AL022076 1778 AF026540 1200 U96128 32514 Z73902 37036 ADOOOO18 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) Mycobacterium tuberculosis H37Rv complete genome; segment 16/162.

Mycobacterium tuberculosis H37Rv complete genome; segment 16/162.

Homo sapiens chromosome 21 cdone LLNLc1 16H-1l124 map 21 q21, SEQUENCING IN PROGRESS in unordered pieces.

Mycobacterium tuberculosis H37Rv complete genome; segment 1571162.

Mycobacterlum tuberculosis Mycobacterium tuberculosis Homo sapiens Mycobacterium tuberculosis Mycobacterium tuberculosis U DP-g alacto pyra nose mutase (gl) gene, complete Mycobacteriumn cds. tuberculosis Mycobacterlum tuberculosis UDP-galactopyranose mutase (gl) gene, complete Mycobacteriumn cds. tuberculosis Mycobacteriumn tuberculosis H37Rv complete genome; segment 59/162. Mycobacterium tuberculosis Mycobacternum tuberculosis sequence from clone yl 51. Mycobacterium tuberculosis Mycobacterium leprae cosmid B1 549. Mycobacterlum leprae Mycobacterium tuberculosis H-37Rv complete genome; segment 59/162. Mycobacterium tuberculosis Mycobacterlum tuberculosis sequence from clone y151. Mycobacteriumn tuberculosis Streptomyces aureofaciens glycogen branching enzyme (glgB) gene, complete Streptomyces; cds. aureofaciens Corynebacterium glutamicum yjcc gene, amtR gene and citE gene, partial. Corynebacterium glutamicum Caenorhabditis elegans cosmid M106, complete sequence. Caenorhabditis elegans UI-R-C3-sz-h-03-0-UI.si UI-R-C3 Rattus norvegicus cODNA clone UI-R-C3-sz-h- Rattus norvegicus 03-0-Ul 3, mRNA sequence.

Mycobacterium tuberculosis H37Rv complete genome; segment 155/162. Mycobacterium tuberculosis Pseudomonas aeruginosa (oriX). glycerol diffusion facilitator (glpF). glycerol Pseudomonas aeruginosa kinase (glpK), and Glp repressor (glpR) genes, complete cds, and (orIK) gene.

partial cds.

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

Arabidopsis thaliana chromosome 1 BAG T1 71-13 sequence, complete Arabidopsis thaliana sequence.

Mus musculus Blk locus, alpha-D-galactosidase A (Ags). ribosomal protein Mus musculus (1-441L), and Bruton's tyrosine kinase (Btk) genes, complete cds.

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

61,677 37,170 19,820 36,957 67,627 70,417 38,532 60,575 57,486 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-1998 25-MAR-1 998 17-Jun.98 10-DEC.1996 29-Sep-94 10-DEC-I1996 25-MAY-i 995 12-Aug-99 2-Sep-99 3-Jul-99 24-Jun-99 18-MAY-1 997 28-Aug.99 5-Jan-98 5-Aug-99 13-Feb-97 01-MAY-I1998 rxa02654 1008 GBBA1:AB015974 GBEST6:N65787 GBPL2:T17H3 GBRO:MMU58105 rxa02666 891 GBPR3:AC04643 2007203042 29 Jun 2007 GBPR3:AC00-4643 43,411 AC004643 G8-BA2:AF049897 9 196 AF049897 Table 4 (continued) Homo sapiens chromosome 16, cosmid clone 363E3 (LANL). complete Homo sapiens sequence.

Corynebacterium glutamicum N-acetylglutamnylphosphate reductase (argC). Corynebacterium ornithine acetyltransterase (argJ), N-acetylglutamate inase (argB), glutamicum acetylornithine transaminase (argD). ornithine carbamoyltransferase (argF).

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

Paracoccus denitrificans NADH dehydrogenase (URF4). (NO08). (NQO9). Paracoccus denitrificans (URF6), (NO010), (NQO1l1), (N0012). (NQO13). and (NQO14) genes, complete cds's: biotin (acetyl-CoA carboxyll ligase (birA) gene, complete Wis.

Mycobacterium tuberculosis H37Rv complete gertome: segment 101/1 62. Mycobacterium 41,599 01-MAY-1998 40.413 1-Jul-96 rxa02675 1980 GB-BA1:PDENQOURF GB GAl :MTCY339 GBBA1:MXADEVRS rxa02694 1065 GB_8A1:BACLDH GBBA1:BACLDHL GBPAT:A06664 ra02729 844 GBESTI5:AA494626 GBEST15:AA494626 10425 42861 L02354 Z77 163 40,735 36,47 1 20-MAY-i1993 17-Jun-96 2452 L19029 Myxococcus xanthus devR and devS genes, complete cds's.

1147 M19394 B.caldolyticus lactate dehydrogenase (LDl-) gene, complete cds.

1361 M14788 B.stearothermophitus Ict gene encoding L-lactate dehydrogenase. complete cds.

1350 A06664 B.stearothemfnophilus Ict gene.

121 ArA494626 faO9dO4.rl Zebrafish lCRFzfls Danio rerio cDNA clone 1 1A22 5' similar to TR:G31171163 G1171163 G/T-MISMATCH BINDING PROTEIN.;, mRNA sequence.

121 AA494626 fa09dO4-rl Zebrafish ICRFzfls Danlo rerio cDNA clone 1 1A22 5 similar to TR:G1 171163 G1 171163 GIT-MISMATCH- BINDING PROTEIN.:., mRNA sequence.

tuberculosis Myxococcus xanthus Bacillus caldolyticus Bacillus stearothermophilus Bacillus slearothermophilus Danio rerio Danio rerio 38,477 27-Jan-94 57,371 26-Apr-93 57.277 26-Apr-93 57,277 29-Jul-93 50.746 27-Jun-97 CA 36,364 27-Jun-97 37.059 29-DEC-i 998 42,149 27-Jun-97 37.655 15-Nov-99 99,560 24-Jun-98 38.363 1 9-Jun-98 rxa02730 1161 GBEST19:AA758660 233 GB EST15:AA494626 121 AA758660 ah67dOG.sl Soares-testisNHT Homo sapiens cDNA clone 1320683 mRNA Homo sapiens sequence.

AA494626 fa09dO4.rl Zebrafish ICRFzfls Danio rerio cDNA clone I 1A22 5' similar to Danjo rerio TR:G1 171163 G1 171163 GIT-MISMATCH BINDING PROTEIN. mRNA sequence.

AC006285 Homo sapiens, complete sequence. Homo sapiens E1 3655 gDNA encoding glucose-6-phosphate dehydrogenase. Corynebacteriu GBPR4:AC00)6285 GBPAT:E13655 150172 2260 m rxa02737 1665 GBBA1:MTCY493 40790 Z95844 Mycobacteriumn tuberculosis H37Rv complete genome: segment 63/162.

rxa02738 1203 rxa02739 2223 GBBA1:SC5A7 GB-PAT:E 13655 GBBA1:SCC22 GBBA1:SC5A7 GBBA1:AB023377 40337 AL031 107 Streptomyces coelicolor cosmid 5A7.

2260 E 13655 gDNA encoding glucose -6-phosphate dehydrogenase.

glutamicumn Mycobacterium tuberculosis Streptomyces coelicolor Corynebacterium glutamicum Slreptomyces coelicolor Streptomyces coelicolor Corynebacterium glutamicumn 39.44 4 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 coelicolor cosmid C22.

Streptomyces coelicolor cosmid 5A7.

Corynebacterium glutamicum IRI gene for lransketolase. complete cds.

2007203042 29 Jun 2007 GBBA1:MLCL536 GBBA1:U00013 rxa02740 1053 GBHTG2:AC006247 GB-HTG2:AC006247 GB HTG3:AC007 150 36224 Z99125 35881 U00013 174368 AC006247 174368 AC006247 121474 AC007150 rxa02741 1089 GBHTG2:AC0O4951 129429 GBHTG2:A0004951 129429 GBIN1:AB006546 931 rxa02743 1161 GSBBA1:MLCL536 36224 GBBAI:U00013 35881 GBHTG2:AC007401 83657 rxa02797 1026 GBBA1:CGBETPGEN 2339 GBGSS9:AQ148714 405 AC00495 1 ACOD4951 AB006546 Z99 125 U00013 AC007401 X93514 AQ148714 Table 4 (continued) Mycobacterium Ieprao cosmld 1536. Mycobacterium leprae 61.573 Mycobacteriumn Ieprae cosmid B1496. Mycobacteriumn leprae 61,573 Drosophila melanogaster chromosome 2 clone BACR48110 (D505) RPCI-98 Drosophila mnelanogaster 37,105 48.1.10 map 49E6-49F8 strain y; cn bw sp, -SEQUENCING IN PROGRESS -,17 unordered pieces.

Drosophila melanogaster chromosome 2 clone BACR48110 (0D505) RPCI-98 Drosophila melanogasler 37,105 48.1.10 map 49E6-49F8 strain y; cn bw sp. SEQUENCING IN PROGRESS 17 unordered pieces.

Drosophila melanogaster chromosome 2 clone 8ACR16PI3 (D597) RPCI-98 Drosophila melanogaster 36.728 16.P.13 map 49E-49F strain y; cn bow sp, -SEQUENCING IN PROGRESS-, 87 unordered pieces.

Homo sapiens clone 0J1022114. SEQUENCING IN PROGRESS ,14 Homo sapiens 33,116 unordered pieces.

Homo sapiens clone DJ1022114, SEQUENCING IN PROGRESS ,14 Homo sapiens 33,116 unordered pieces.

Ephydatia Ituviatilis mRNA for G protein a subunit 4, partial cds. Ephydatia fluviatilis 36,379 Mycobacteriumn leprae cosmid L536. Mycobacterium Ieprae 48,401 Mycobacterium leprae cosmid B1496. Mycobacterium Ieprae 48,401 Homo sapiens clone NHO5O1007, -SEQUENCING IN PROGRESS-. 3 Homo sapiens 37,128 unordered pieces.

C.glutamicum betP gene. Corynebacterium 38.889 glutamicum HS_3136_AlA03_MR cir Approved Human Genomic Sperm Library D Homo Homo sapiens 34,321 sapiens gonomIc clone Plate='31 36 Cot=5 Row=A, genomic survey sequence.

Bacillus firmus dppABC operon. dipeptIde transporter protein dppA gene. Bacillus firmus 38.072 partial ods, and dipeptide transporter proteins dppB and dppC genes, complete cds.

Mycobacterium Ieprae cosmid B229. Mycobacleriumn Ieprae 34,462 Pseudomonas syringae pv. sytingae putative dihydropteroate synthase gene, Pseudomonas syrtngae 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 coelicolor 59,314 Homo sapiens chromosome 17 clone 2020_-K_-17 map 17, -SEQUENCING H-omo sapiens 37,607 IN PROGRESS 12 unordered pieces.

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

04-DEC- 1998 01-MAR-1994 2-Aug-99 2-Aug.99 20-Sep.99 12-Jun-98 12-Jun-98 23-Jun-99 04-DEC-1998 01-MAR-1994 26-Jun-99 8-Sep-97 08-OCT-i1998 rxa02803 680 rxa02821 363 GBBA1:BFU64514 GB-BA1 :U00020 GBBA2:P5U85643 GBBA1:5C6G4 GB-HTG2:ACOO81 05 GB-HTG2:ACOO81 05 3837 U64514 1-Feb-97 36947 4032 41055 91421 91421 U00020 U85643 AL031317 AC0081 05 AC008105 01-MAR-1994 9-Apr-97 20-Aug-98 22-Jul-99 22-Jul-99 GB-EST33:AV1 17143 222 AVI 17143 AVI 17143 Mus muscutus C57BtJ6J 10-day embryo Mus musculus cONA clone Mus musculus 2610200J 17, mRNA sequence.

40.157 30-Jun-99 2007203042 29 Jun 2007 ra02829 373 GB-HTG1:HSU9G8 GBHTG1:HSU9G8 GBPR3:HSU8565 rxc03216 1141 GB-TG3:AC008184 48735 48735 39550 151720 AL008714 AL008714 Z69724 AC008 184 GB-EST15:AA477537 411 AA477537 Table 4 (continued) Homo sapiens chromosome X clone LLOXNCO1-9G8, SEQUENCING IN Homo sapiens PROGRESS -,in unordered pieces.

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

Human DNA sequence from cosmid U85B35. between mlarkers DXS366 and Homo sapiens DX S87 on chromosome X.

Drosophila melanogaster chromosome 2 clone BACRO4005 (D540) RPCI-98 Drosophila melanogasler 04.0.5 map 36E5-36F2 strain y; cn bw sp. SEQUENCING IN PROGRESS unordered pieces.

zu36912.rl Soares ovary tumor NbHOT H-omo sapiens cDNA clone Homo sapiens IMAGE:740134 5'similar to contains Alu repetitive element:contalns element HGR repetitive element mRNA sequence.

fagldOS.yl zebrafish fin dayll regeneration Danio rerio cONA mRNA Danjo rerlo sequence- Streplomyces coeticolor cosmld 3F9. Streptomyces coellcolor A3(2) SlIincolnensis (78-11) Lincomycin production genes. Streptomyces lincolnensis Homo sapiens chromosome 15 clone RP1 1-424J10 map SEQUENCING Homo sapiens IN PROGRESS 41 unordered pieces.

Homo sapiens chromosome 19. cosmid R30217. complete sequence. Homo sapiens Spombe chromosome I cosmid c926. Schizosaccharomyces pombe Archaeoglobus fulgidus section 26 of 172 of the complete genome. Archaeoglobus fulgidlus 41. 595 4 1.595 4 1.595 39,600 23-Nov-99 23-Nov-99 23-Nov-99 2-Aug-99 37,260 9-Nov-97 GBEST26:A1330662 rnxs03215 1038 GBDBAJ:SC3F9 G8 BAt :SLLI NC GB-HTG5:AC009660 rxs03224 1288 GBPR3:AC004076 GBPL2:5PAC926 GB_8A2:AE001081 412 19830 36270 204320 41322 23193 11473 A1330662 AL023862 X79146 AC009660 AC004 076 ALl110469 AE001081 37.805 48.657 39,430 35,151 37,788 38.474 35.87 1 28-DEC-1998 10-Feb-99 15-MAY-1996 04-DEC-1999 29-Jan-98 2-Sep-99 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 0 C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I of the original volume of the culture all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/1 sucrose, 2.46 g/1 MgSO, x 7H 2 0, 10 ml/1 KH,PO, solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/1 M12 concentrate (10 g/1 (NH) 2 I g/l NaCI, 2 g/1 MgSO, x 7H 2

O,

0.2 g/1 CaCI,, 0.5 g/l yeast extract (Difco), 10 ml/l trace-elements-mix (200 mg/1 FeSO.

x HO 2 10 mg/1 ZnSO, x 7 HO 2 3 mg/1 MnCl, x 4 HO, 30 mg/l H,BO, 20 mg/l CoCI, x 6 HO, 1 mg/1 NiCI, x 6 H 2 O, 3 mg/1 NaMoO, x 2 HO, 500 mg/l 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/1 nicotinic acid, 40 mg/l pyridoxole hydrochloride, 200 mg/1 myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37'C, the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HC1, 1 mM EDTA, pH The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution and 0.5 ml NaCI solution (5 M) are added. After adding ofproteinase K to a final concentration of 200 pg/ml, the suspension is incubated for ca. 18 h at 37 0 C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroformisoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20 0 C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing ptg/ml RNaseA and dialysed at 4°C against 1000 ml TE-buffer for at least 3 hours.

During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added. After a 119 min incubation at -20 0 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 pHM1519 or pBLI) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley Sons) to which a origin or replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E.

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

Using standard methods, it is possible to clone a gene of interest into one of the 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 Schafer, A et al.

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

Genes may be overexpressed in C. glutamicum strains using plasmids which comprise 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 -123advantageous to supply mixtures of different carbon sources. Other possible carbon Ssources 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 NHCI or NH,OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast r extract, meat extract and others.

SInorganic salt compounds which may be included in the media include the Schloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, C, 10 molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook "Applied Microbiol. Physiology, A Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.

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

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

An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It 124is also possible to maintain a constant culture pH through the addition of NaOH or 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 6 oo of 0.5 1.5 using cells grown on agar plates, such as CM plates (10 g/l glucose, 2,5 g/l NaCI, 2 g/1 urea, 10 g/1 polypeptone, 5 g/l 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 125found, for example, in the following references: Dixon, and Webb, (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.

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

Press: Oxford; Boyer, ed. (1983) The Enzymes, 3 rd ed. Academic Press: New York; Bisswanger, (1994) Enzymkinetik, 2 n d ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, Bergmeyer, Grafl, 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. el al. (1995) EMBO J. 14: 3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.

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

Example 9: Analysis of Impact of Mutant Protein on the Production of the Desired Product The effect of the genetic modification in C. glutamicum on production of a desired compound (such as an amino acid) can be assessed by growing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product an amino acid). Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, 126- Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm el al.

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

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

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

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

The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography

(HPLC),

spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27- 32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540- 547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. el 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. Nail. Acad. Sci. USA 128- 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score 100, wordlength 12 to obtain nucleotide sequences homologous to SMP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score 50, wordlength 3 to obtain amino acid sequences homologous to SMP protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program XBLAST and NBLAST) for the specific sequence being analyzed.

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

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

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

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

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

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

(1997) Science 278: 680-686).

DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label -130may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or-absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).

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

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

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

Hybridization of labeled nucleic acids to microarrays is described in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. (1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for example, as 131 described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).

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

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

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

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

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

(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 132- 1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art.

Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors 35 S-methionine, 3S-cysteine, 4 C-labelled amino acids, 15 N-amino acids, "1NO 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, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals.

133

EQUIVALENTS

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

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

Claims (30)

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:245, or a complement thereof.

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:246, 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:246, 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:245, 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:245, 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:246, 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. I 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:246.

17. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:246.

18. An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:245.

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:246. An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:246, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID NO:246. 136

21. An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:245.

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

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

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

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

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

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

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

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

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

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

33. A host cell comprising the nucleic acid molecule of SEQ ID NO:245, wherein the nucleic acid molecule is disrupted.

34. A host cell comprising the nucleic acid molecule of SEQ ID NO:245, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:245. A host cell comprising the nucleic acid molecule of SEQ ID NO:245, 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 P20679AU06