Gene conferring lysozyme insensitivity to corynebacterium
View Patent ↗The present invention relates to a protein having an activity of giving a lysozyme insensitivity to a lysozyme-sensitive microorganism belonging to Corynebacterium glutamicum ; DNA which codes for the protein; a recombinant vector containing the DNA; a transformant obtained by introducing the recombinant vector into a host cell; a bacterium having a lysozyme sensitivity in which the activity of the protein is inactivated; and a method for producing an amino acid using the bacterium.
1. An isolated DNA encoding a protein which comprises the amino acid sequence of SEQ ID NO: 2, and confers to a Corynebacterium glutamicum microorganism an ability to grow in a medium containing 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 20 μg/ml thiamine and 100 μg/ml lysozyme.
2. An isolated DNA comprising the nucleotide sequence of SEQ ID NO: 1; or a DNA hybridizing with the DNA having a complementary nucleotide sequence of SEQ ID NO: 1 at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride and encoding a protein which confers to a Corynebacterium glutamicum microorganism an ability to grow in a medium containing 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 20 μg/ml thiamine and 100 μg/ml lysozyme, wherein the hybridization further includes a step of washing under the condition of 65° C. by the use of solution containing 15 to 300 mM sodium chloride and 1.5 to 30 mM sodium citrate.
3. An isolated DNA which is contained in a plasmid carried by FERM BP-6479 and codes for a protein which confers to a Corynebacterium glutamicum microorganism an ability to grow in a medium containing 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 20 μg/ml thiamine and 100 μg/ml lysozyme.
4. The DNA according to claim 1 , wherein the microorganism is a mutant strain of Corynebacterium glutamicum which cannot grow in a medium containing 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 20 μg/ml thiamine and 100 μg/ml lysozyme prior to transformation with said DNA.
5. The DNA according to claim 2 , wherein the microorganism is a mutant strain of Corynebacterium glutamicum which cannot grow in a medium containing 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 20 μg/ml thiamine and 50 μg/ml lysozyme prior to transformation with said DNA.
6. The DNA according to claim 3 , wherein the microorganism is a mutant strain of Corynebacterium glutamicum that cannot grow in a medium containing 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 20 μg/ml thiamine and 50 μg/ml lysozyme prior to transformation with said DNA.
7. The DNA according to claim 1 , wherein the DNA is a DNA derived from a microorganism belonging to the genus Corynebacterium.
8. The DNA according to claim 2 , wherein the DNA is a DNA derived from a microorganism belonging to the genus Corynebacterium.
9. The DNA according to claim 3 , wherein the DNA is a DNA derived from a microorganism belonging to the genus Corynebacterium.
10. The DNA according to claim 1 , wherein the DNA is a DNA derived from a microorganism belonging to Corynebacterium glutamicum.
11. The DNA according to claim 2 , wherein the DNA is a DNA derived from a microorganism belonging to Corynebacterium glutamicum.
12. The DNA according to claim 3 , wherein the DNA is a DNA derived from a microorganism belonging to Corynebacterium glutamicum.
13. A recombinant vector comprising the DNA according to any one of claims 1 , 4 , 7 , and 10 .
14. A recombinant vector comprising the DNA according to any one of claims 2 , 5 , 8 , and 11 .
15. A recombinant vector comprising the DNA according to any one of claims 3 , 6 , 9 , and 12 .
16. A transformant prepared by introducing the recombinant vector of claim 13 into a host cell.
17. A transformant prepared by introducing the recombinant vector of claim 14 into a host cell.
18. A transformant prepared by introducing the recombinant vector of claim 15 into a host cell.
19. A method for producing a protein, which comprises culturing in a medium a transformant prepared by introducing a recombinant vector comprising DNA according to any one of claims 1 , 4 , 7 , and 10 into a host cell, producing and accumulating the protein encoded by the DNA in the culture, and collecting the protein from the culture.
20. A method for producing a protein, which comprises culturing in a medium a transformant prepared by introducing a recombinant vector comprising DNA according to any one of claims 2 , 5 , 8 and 11 into a host cell, producing and accumulating the protein encoded by the DNA in the culture, and collecting the protein from the culture.
21. A method for producing a protein, which comprises culturing in a medium a transformant prepared by introducing a recombinant vector comprising DNA according to any one of claims 3 , 6 , 9 and 12 into a host cell, producing and accumulating the protein encoded by the DNA in the culture, and collecting the protein from the culture.
22. A DNA fragment which comprises a nucleotide sequence of the nucleotide position numbers 271 to 1593 in the nucleotide sequence identified as SEQ ID NO: 1.
23. A recombinant vector comprising the DNA according to claim 22 .
24. A transformant prepared by introducing the recombinant vector of claim 23 into a host cell.
25. A method for producing a protein, which comprises culturing in a medium a transformant prepared by introducing a recombinant vector comprising DNA according to claim 22 into a host cell, producing and accumulating the protein encoded by the DNA in the culture, and collecting the protein from the culture.