Glucose-6-phosphate dehydrogenase
View Patent ↗The present invention relates to a novel glucose-6-phosphate dehydrogenase (hereinafter referred to as “G6PD”) derived from a bacterium belonging to the genus Corynebacterium , a DNA encoding the enzyme, a recombinant DNA comprising the DNA, a transformant comprising the recombinant DNA, a transformant comprising the DNA on its chromosome, and a process for producing L-amino acid or G6PD which comprises culturing the transformant. According to the present invention, a modified G6PD and a DNA encoding the G6PD are obtained, and the productivity of L-amino acid by a microorganism can be improved by using the modified G6PD.
1. An isolated or purified DNA which encodes a polypeptide selected from the group consisting of (a) to (d):
(a) a polypeptide which consists of SEQ ID NO:2,
(b) a polypeptide which consists of a variant of SEQ ID NO:2, wherein only Ala at position 213 in SEQ ID NO:2 is replaced with another amino acid, and a variant of SEQ ID NO:2, wherein glucose-6-phosphate dehydrogenase activity,
(c) a polypeptide which consists of SEQ ID NO:12, and
(d) a polypeptide which consists of a variant of SEQ ID NO:12, wherein up to ten amino acids other than the amino acid residue at position 213 in SEQ ID NO:12 are deleted, substituted or added, and a variant of SEQ ID NO:12, has glucose-6-phosphate dehydrogenase activity.
2. An isolated or purified DNA which consists of the nucleotide sequence SEQ ID NO:1.
3. An isolated or purified DNA which consists of a variant of SEQ ID NO:1, wherein only positions 637 to 639 in the nucleotide sequence of SEQ ID NO:1 is replaced with a codon encoding an amino acid other than Ala and wherein said variant encodes a polypeptide with glucose-6-phospahte dehydrogenase activity.
4. An isolated or purified DNA which consists of the nucleotide sequence SEQ ID NO:11.
5. An isolated or purified DNA which hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO:1 at 65° C. in the presence of 0.7 to 1.0 mol/l of sodium chloride using a filter on which colony- or plaque-derived DNA is immobilized, followed by washing at 65° C. with 0.1 to 2×SSC solution, and encodes a polypeptide having glucose-6-phosphate dehydrogenase activity,
wherein a nucleotide sequence corresponding to the nucleotide sequence of positions 637 to 639 encoding in the nucleotide sequence SEQ ID NO:1 is replaced with a codon encoding an amino acid other than Ala and an amino acid sequence of the polypeptide encoded by the hybridizable DNA has homology of 95% or more compared with the amino acid sequence of SEQ ID NO:12.
6. An isolated or purified DNA which hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO:1 at 65° C. in the presence of 0.7 to 1.0 mol/l of sodium chloride using a filter on which colony- or plaque-derived DNA is immobilized, followed by washing at 65° C. with 0.1 to 2×SSC solution, and encodes a polypeptide having glucose-6-phosphate dehydrogenase activity,
wherein a nucleotide sequence corresponding to the nucleotide of position 637 in the nucleotide sequence SEQ ID NO:1 is replaced with adenine and an amino acid sequence of the polypeptide encoded by the hybridizable DNA has homology of 95% or more compared with the amino acid sequence of SEQ ID NO:12.
7. A recombinant DNA which is obtained by inserting the DNA according to any one of claims 1 to 6 into a vector.
8. The recombinant DNA according to claim 7 , wherein the recombinant DNA is replicates in a microorganism belonging to the genus Escherichia or the genus Corynebacterium.
9. A plasmid pCRBzwfM found in Escherichia coli TOP10 (FERM BP-7135).
10. A transformant which is obtained by introducing the recombinant DNA according to claim 8 into a host cell.
11. The transformant according to claim 10 , wherein the host cell is a microorganism which produces L-amino acids.
12. The transformant according to claim 11 , wherein the host cell belongs to the genus Escherichia or the genus Corynebacterium.
13. A transformant belonging to the genus Escherichia or the genus Corynebacterium , which comprises a genome into which the DNA according to any one of claims 3 to 6 is integrated.
14. The transformant according to claim 13 , wherein the host cell is Corynebacterium glutamicum.
15. A process for producing a polypeptide, which comprises culturing the transformant according to claim 11 in a medium to form and accumulate in culture a polypeptide selected from the group consisting of (a) to (d):
(a) a polypeptide which consists of SEQ ID NO:2,
(b) a polypeptide which consists of a variant of SEQ ID NO:2, wherein only Ala at position 213 in SEQ ID NO:2 is replaced with another amino acid, and said variant of SEQ ID NO:2 has glucose-6-phosphate dehydrogenase activity,
(c) a polypeptide which consists of SEQ ID NO:12, and
(d) a polypeptide which consists of a variant of SEQ ID NO:12, wherein up to ten amino acids other than the amino acid residue at position 213 in SEQ ID NO:12 are deleted, substituted or added, and said variant of SEQ ID NO:12 has glucose-6-phosphate dehydrogenase activity, and
recovering the polypeptide from the culture.
16. A process for producing L-lysine, L-threonine, L-isoleucine, L-tryptophan, L-phenylalanine, L-tyrosine, L-histidine or L-cysteine, which comprises culturing the transformant according to claim 11 in a medium to form and accumulate the L-amino acid which is biosynthesized using NADPH in the culture and said recombinant DNA or transformant and recovering the L-amino acid from the culture.
17. The process for producing L-amino acid according to claim 16 , wherein the L-amino acid is L-lysine.