Corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins

Abstract

Isolated nucleic acid molecules, designated SRT nucleic acid molecules, which encode novel SRT proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing SRT nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated SRT proteins, mutated SRT proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of SRT genes in this organism.

1 . An isolated nucleic acid molecule from  Corynebacterium glutamicum  encoding a stress, resistance, or tolerance gene, or a portion thereof, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.  
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said stress, resistance, or tolerance gene is selected from the group consisting of nucleic acid molecules involved in a stress response, tolerance, or resistance to temperature stresses, pH stresses, oxygen stresses, osmotic stresses, toxic chemicals, oxygen radicals, antibiotics, or to lincomycin.  
     
     
         3 . An isolated  Corynebacterium glutamicum  nucleic acid molecule selected from the group consisting of those sequences set forth in Appendix A, or a portion thereof, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.  
     
     
         4 . An isolated nucleic acid molecule which encodes a polypeptide sequence selected from the group consisting of those sequences set forth in Appendix B, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.  
     
     
         5 . An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide selected from the group of amino acid sequences consisting of those sequences set forth in Appendix B, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.  
     
     
         6 . An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% homologous to a nucleotide sequence selected from the group consisting of those sequences set forth in Appendix A, or a portion thereof, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.  
     
     
         7 . An isolated nucleic acid molecule comprising a fragment of at least 15 nucleotides of a nucleic acid comprising a nucleotide sequence selected from the group consisting of those sequences set forth in Appendix A, provided that the nucleic acid molecule does not consist of any of the F-designated genes set forth in Table 1.  
     
     
         8 . An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule of any one of claims  1 - 7  under stringent conditions.  
     
     
         9 . An isolated nucleic acid molecule comprising the nucleic acid molecule of  claim 1  or a portion thereof and a nucleotide sequence encoding a heterologous polypeptide.  
     
     
         10 . A vector comprising the nucleic acid molecule of  claim 1 .  
     
     
         11 . The vector of  claim 10 , which is an expression vector.  
     
     
         12 . A host cell transfected with the expression vector of  claim 11 .  
     
     
         13 . The host cell of  claim 12 , wherein said cell is a microorganism.  
     
     
         14 . The host cell of  claim 13 , wherein said cell belongs to the genus  Corynebacterium  or  Brevibacterium.    
     
     
         15 . The host cell of claim. 12, wherein the expression of said nucleic acid molecule results in the modulation in production of a fine chemical from said cell.  
     
     
         16 . The host cell of  claim 15 , 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.  
     
     
         17 . A method of producing a polypeptide comprising culturing the host cell of  claim 12  in an appropriate culture medium to, thereby, produce the polypeptide.  
     
     
         18 . An isolated stress, resistance, or tolerance polypeptide from  Corynebacterium glutamicum , or a portion thereof.  
     
     
         19 . The protein of  claim 18 , wherein said stress, resistance, or tolerance polypeptide is selected from the group consisting of proteins involved in a stress response, tolerance, or resistance to temperature stresses, pH stresses, oxygen stresses, osmotic stresses, toxic chemicals, oxygen radicals, antibiotics, or to lincomycin.  
     
     
         20 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of those sequences set forth in Appendix B, provided that the amino acid sequence is not encoded by any of the F-designated genes set forth in Table 1.  
     
     
         21 . An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising an amino acid sequence selected from the group consisting of those sequences set forth in Appendix B, or a portion thereof, provided that the amino acid sequence is not encoded by any of the F-designated genes set forth in Table 1.  
     
     
         22 . The isolated polypeptide of  claim 18 , further comprising heterologous amino acid sequences.  
     
     
         23 . An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% homologous to a nucleic acid selected from the group consisting of those sequences set forth in Appendix A, provided that the nucleic acid molecule does not consist of any of the F-designated nucleic acid molecules set forth in Table 1.  
     
     
         24 . An isolated polypeptide comprising an amino acid sequence which is at least 50% homologous to an amino acid sequence selected from the group consisting of those sequences set forth in Appendix B, provided that the amino acid sequence is not encoded by any of the F-designated genes set forth in Table 1.  
     
     
         25 . A method for producing a fine chemical, comprising culturing a cell containing a vector of  claim 12  such that the fine chemical is produced.  
     
     
         26 . The method of  claim 25 , wherein said method further comprises the step of recovering the fine chemical from said culture.  
     
     
         27 . The method of  claim 25 , wherein said method further comprises the step of transfecting said cell with the vector of  claim 11  to result in a cell containing said vector.  
     
     
         28 . The method of  claim 25 , wherein said cell belongs to the genus  Corynebacterium  or  Brevibacterium.    
     
     
         29 . The method of  claim 25 , 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 ketosoreduclum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum , and those strains set forth in Table 3.  
     
     
         30 . The method of  claim 25 , wherein expression of the nucleic acid molecule from said vector results in modulation of production of said fine chemical.  
     
     
         31 . The method of  claim 25 , 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.  
     
     
         32 . The method of  claim 25 , wherein said fine chemical is an amino acid.  
     
     
         33 . The method of  claim 32 , wherein said amino acid is drawn from the group consisting of: lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine, and tryptophan.  
     
     
         34 . A method for producing a fine chemical, comprising culturing a cell whose genomic DNA has been altered by the inclusion of a nucleic acid molecule of any one of claims  1 - 9 .  
     
     
         35 . A method for diagnosing the presence or activity of  Corynebacterium diphtheriae  in a subject, comprising detecting the presence of one or more of the sequences set forth in Appendix A or Appendix B in the subject, provided that the sequences are not or are not encoded by any of the F-designated sequences set forth in Table 1, thereby diagnosing the presence or activity of  Corynebacterium diphtheriae  in the subject.  
     
     
         36 . A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth in Appendix A, wherein the nucleic acid molecule is disrupted.  
     
     
         37 . A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth in Appendix A, wherein the nucleic acid molecule comprises one or more nucleic acid modifications from the sequence set forth in Appendix A.  
     
     
         38 . A host cell comprising a nucleic acid molecule selected from the group consisting of the nucleic acid molecules set forth in Appendix A, wherein the regulatory region of the nucleic acid molecule is modified relative to the wild-type regulatory region of the molecule.