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Corynebacterium glutamicum genes encoding novel proteins

Corynebacterium glutamicum genes encoding novel proteins description/claims

This application is a continuation of U.S. application Ser. No. 11/092,052, filed Mar. 28, 2005 which is a continuation of U.S. application Ser. No. 09/605,703, filed Jun. 27, 2000, which claims priority to U.S. Provisional Patent Application Ser. No. 60/142,764, filed Jul. 8, 1999, and U.S. Provisional Patent Application Ser. No. 60/152,318, filed Sep. 3, 1999. The entire contents of each of these aforementioned applications are hereby expressly incorporated herein by reference.

This application incorporates herein by reference the material contained on the compact disks submitted herewith as part of this application. Specifically, the file “Sequence Listing.txt” (8.19 MB) contained on each of Copy 1, Copy 2 and the CRF copy of the Sequence Listing is hereby incorporated herein by reference. Each of these files were created on Jan. 25, 2007. In addition the files “Appendix A.txt” (1.33 MB) and “Appendix B.txt” (478 KB) contained on the compact disks entitled “Appendices Copy 1” and “Appendices Copy 2” are hereby incorporated herein by reference. Each of these files were created on Jan. 28, 2007.

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.

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 marker and fine chemical production (MCP) 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 MCP nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the MCP nucleic acids of the invention, or modification of the sequence of the MCP nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).

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

The MCP 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 MCP proteins encoded by the novel nucleic acid molecules of the invention may be involved, for example, in the direct or indirect production of one or more fine chemicals from C. glutamicum. The MCP proteins of the invention may also participate in the degradation of hydrocarbons or the oxidation of terpenoids. These proteins may also be utilized for the identification of Corynebacterium glutamicum or organisms related to C. glutamicum; the presence of an MCP protein specific to C. glutamicum and related species in a mixture of proteins may indicate the presence of one of these bacteria in the sample. Further, these MCP proteins may have homologues in plants or animals which are involved in a disease state or condition; these proteins thus may serve as useful pharmaceutical targets for drug screening and the development of therapeutic compounds.

Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to modulate the production of one or more fine chemicals. This modulation 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. For example, by modifying the activity of a protein involved in the biosynthesis or degradation of a fine chemical (i.e., through mutagenesis of the corresponding gene), one may directly modulate the ability of the cell to synthesize or to degrade this compound, thereby modulating the yield and/or efficiency of production of the fine chemical. Similarly, by modulating the activity of a protein which regulates a fine chemical metabolic pathway, one may directly influence whether the production of the desired compound is up- or down-regulated, either of which will modulate the yield or efficiency of production of the fine chemical from the cell.

Indirect modulation of fine chemical production may also result by modifying the activity of a protein of the invention (i.e., by mutagenesis of the corresponding gene) such that the overall ability of the cell to grow and divide or to remain viable and productive is increased. The production of fine chemicals from C. glutamicum is generally accomplished by the large-scale fermentative culture of these microorganisms, conditions which are frequently suboptimal for growth and cell division. By engineering a protein of the invention (e.g., a stress response protein, a cell wall protein, or proteins involved in the metabolism of compounds necessary for cell growth and division to occur, such as nucleotides and amino acids) such that it is better able to survive, grow, and multiply in such conditions, it may be possible to increase the number and productivity of such engineered C. glutamicum cells in large-scale culture, which in turn should result in increased yields and/or efficiency of production of one or more desired fine chemicals. Further, the metabolic pathways of any cell are necessarily interrelated and coregulated. By altering the activity or regulation of any one metabolic pathway in C. glutamicum (i.e., by altering the activity of one of the proteins of the invention which participates in such a pathway), it is possible to concomitantly alter the activity or regulation of other metabolic pathways in this microorganism, which may be directly involved in the synthesis or degradation of a fine chemical.

The invention provides novel nucleic acid molecules which encode proteins, referred to herein as MCP proteins, which are capable of, for example, modulating the production or efficiency of production of one or more fine chemicals from C. glutamicum, or of serving as identifying markers for C. glutamicum or related organisms. Nucleic acid molecules encoding an MCP protein are referred to herein as MCP nucleic acid molecules. In a preferred embodiment, the MCP protein is capable of modulating the production or efficiency of production of one or more fine chemicals from C. glutamicum, or of serving as identifying markers for C. glutamicum or related organisms. Examples of such proteins include those encoded by the genes set forth in Table 1.

Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an MCP protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of MCP-encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth in Appendix A 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 in Appendix A, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth in Appendix B. The preferred MCP proteins of the present invention also preferably possess at least one of the MCP 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 Appendix B, e.g., sufficiently homologous to an amino acid sequence of Appendix B such that the protein or portion thereof maintains an MCP activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to modulate the production or efficiency of production of one or more fine chemicals from C. glutamicum, or of serving as an identifying marker for C. glutamicum or related organisms. 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 Appendix B (e.g., an entire amino acid sequence selected from those sequences set forth in Appendix B). In another preferred embodiment, the protein is a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of Appendix B (encoded by an open reading frame shown in Appendix A).

In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an MCP 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 Appendix B and is able to modulate the yield, production, and/or efficiency of production of one or more fine chemicals from C. glutamicum, to degrade hydrocarbons, to oxidize terpenoids, to serve as a target for drug development, or to serve as an identifying marker for C. glutamicum or related organisms, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.

In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of Appendix 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 MCP protein, or a biologically active portion thereof.

Another aspect of the invention pertains to vectors, e.g., 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 MCP protein by culturing the host cell in a suitable medium. The MCP protein can then be isolated from the medium or the host cell.

Yet another aspect of the invention pertains to a genetically altered microorganism in which an MCP 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 MCP sequence as a transgene. In another embodiment, an endogenous MCP gene within the genome of the microorganism has been altered, e.g., functionally disrupted, by homologous recombination with an altered MCP gene. In another embodiment, an endogenous or introduced MCP gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional MCP protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an MCP gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the MCP 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.

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