Patent
Isolated nucleic acid molecules, designated MP nucleic acid molecules, which encode novel MP proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing MP nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated MP proteins, mutated MP proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of MP genes in this organism.
1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, or a full complement thereof.
2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a full complement thereof.
3. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the entire nucleotide sequence of SEQ ID NO:1, wherein the nucleic acid molecule encodes a polypeptide having a diaminopimelate epimerase activity, or a full complement thereof.
4. An isolated nucleic acid molecule consisting of a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, or a full complement thereof.
5. An isolated nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, wherein the nucleic acid molecule encodes a polypeptide having a diaminopimelate epimerase activity, or a full complement thereof.
6. An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 95% identical to the entire amino acid sequence of SEQ ID NO:2, wherein the polypeptide has a diaminopimelate epimerase activity, or a full complement thereof.
7. An isolated nucleic acid molecule which hybridizes to the complement of the nucleotide sequence of SEQ ID NO:1 in 6X sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65° C., wherein said nucleic acid molecule encodes a polypeptide with diaminopimelate epimerase activity, or a full complement thereof.
8. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , and 7 and a nucleotide sequence encoding a heterologous polypeptide.
9. A vector comprising the nucleic acid molecule of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , and 7 .
10. The vector of claim 9 , which is an expression vector.
11. A host cell transfected with the expression vector of claim 10 .
12. The host cell of claim 11 , wherein said cell is a microorganism.
13. The host cell of claim 12 , wherein said cell belongs to the genus Corynebacterium or Brevibacterium.
14. A method of producing a polypeptide comprising culturing the host cell of claim 11 in an appropriate culture medium to, thereby, produce the polypeptide.
15. A method for producing an amino acid, comprising culturing the cell of claim 11 such that the amino acid is produced, wherein said cell is a bacterial cell.
16. The method of claim 15 , wherein said method further comprises the step of recovering the amino acid from said culture.
17. The method of claim 15 , wherein said cell belongs to the genus Corynebacterium or Brevibacterium.
18. The method of claim 15 , 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 set forth in Table 3.
19. The method of claim 15 , wherein expression of the nucleic acid molecule from said vector results in modulation of production of said amino acid.
20. The method of claim 15 , wherein said amino acid is a proteinogenic or nonproteinogenic amino acid.
21. The method of claim 15 , wherein said amino acid is a proteinogenic amino acid.
22. The method of claim 15 , 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.
23. A method for producing an amino acid, comprising culturing a bacterial cell whose genomic DNA has been altered by the inclusion of a nucleic acid molecule of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , and 7 .
24. A method for diagnosing the presence or activity of Corynebacterium diphtheriae in a subject, comprising detecting the presence or activity of the nucleic acid molecule of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , and 7 , thereby diagnosing the presence or activity of Corynebacterium diphtheriae in the subject.