Gene variants coding for proteins from the metabolic pathway of fine chemicals
View Patent ↗The present invention relates to mutated nucleic acids and proteins of the metabolic pathway of fine chemicals, to processes for preparing genetically modified producer organisms, to processes for preparing fine chemicals by culturing genetically modified organisms, and to genetically modified organisms themselves.
1. An isolated nucleic acid molecule encoding a transketolase selected from the group consisting of:
a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 70, wherein (i) the amino acid residue at position 75 of SEQ ID NO: 70 is any amino acid except asparagine, (ii) the amino acid residue at position 332 of SEQ ID NO: 70 is any amino acid except alanine, or (iii) the amino acid residue at position 556 of SEQ ID NO: 70 is any amino acid except valine, or a full complement thereof; and
b) an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 69, wherein the nucleic acid molecule comprises one or more nucleic acid modifications at (i) nucleotide residues 223-225 of SEQ ID NO: 69 such that nucleotide residues 223-225 of SEQ ID NO: 69 encode any amino acid except asparagine, (ii) nucleotide residues 994-996 of SEQ ID NO: 69 such that nucleotide residues 994-996 of SEQ ID NO: 69 encode any amino acid except alanine, or (iii) nucleotide residues 1666-1668 of SEQ ID NO: 69 such that nucleotide residues 1666-1668 of SEQ ID NO: 69 encode any amino acid except valine, or a full complement thereof.
2. An isolated nucleic acid construct, comprising at least one nucleic acid molecule of claim 1 .
3. The nucleic acid construct of claim 2 , comprising at least one promoter and a terminator.
4. A method for preparing an isolated transformed host cell comprising introducing into the host cell at least one of
a) the nucleic acid molecule of claim 1 ; or
b) a promoter which is heterologous to an endogenous nucleic acid molecule and which enables the expression of said endogenous nucleic acid molecule, wherein said nucleic acid molecule is selected from the group consisting of:
i) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 70, wherein either (a) the amino acid residue at position 75 of SEQ ID NO: 70 is any amino acid except asparagine, (b) the amino acid residue at position 332 of SEQ ID NO: 70 is any amino acid except alanine, or (c) the amino acid residue at position 556 of SEQ ID NO: 70 is any amino acid except valine, or a full complement thereof; and
ii) an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 69, wherein the nucleic acid molecule comprises one or more nucleic acid modifications at either (a) nucleotide residues 223-225 of SEQ ID NO: 69 such that nucleotide residues 223-225 of SEQ ID NO: 69 encode any amino acid except asparagine, (b) nucleotide residues 994-996 of SEQ ID NO: 69 such that nucleotide residues 994-996 of SEQ ID NO: 69 encode any amino acid except alanine, or (c) nucleotide residues 1666-1668 of SEQ ID NO: 69 such that nucleotide residues 1666-1668 of SEQ ID NO: 69 encode any amino acid except valine, or a full complement thereof.
5. The method of claim 4 , wherein the nucleic acid molecule of a), or the promoter of b), is introduced into a replicating plasmid or is integrated chromosomally.
6. The method of claim 4 , wherein the promoter of b) is functionally linked to the endogenous nucleic acid molecule.
7. An isolated transformed host cell, transformed with at least one of
a) the nucleic acid molecule of claim 1 ; or
c) a promoter which is heterologous to an endogenous nucleic acid molecule and which enables the expression of said endogenous nucleic acid molecule, wherein said nucleic acid molecule is selected from the group consisting of:
i) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO: 70, wherein either (a) the amino acid residue at position 75 of SEQ ID NO: 70 is any amino acid except asparagine, (b) the amino acid residue at position 332 of SEQ ID NO: 70 is any amino acid except alanine, or (c) the amino acid residue at position 556 of SEQ ID NO: 70 is any amino acid except valine, or a full complement thereof; and
ii) an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 69, wherein the nucleic acid molecule comprises one or more nucleic acid modifications at either (a) nucleotide residues 223-225 of SEQ ID NO: 69 such that nucleotide residues 223-225 of SEQ ID NO: 69 encode any amino acid except asparagine, (b) nucleotide residues 994-996 of SEQ ID NO: 69 such that nucleotide residues 994-996 of SEQ ID NO: 69 encode any amino acid except alanine, or (c) nucleotide residues 1666-1668 of SEQ ID NO: 69 such that nucleotide residues 1666-1668 of SEQ ID NO: 69 encode any amino acid except valine, or a full complement thereof.
8. The isolated transformed host cell of claim 7 , wherein the organism is capable of producing an amino acid.
9. A method for the preparation of an amino acid, comprising culturing the isolated transformed host cell of claim 7 in a culturing medium under conditions suitable for the preparation of an amino acid.
10. The method of claim 9 , wherein the amino acid is isolated from the isolated transformed host cell and/or the culturing medium.
11. The method of claim 9 , wherein the isolated transformed host cell is a microorganism.
12. The method of claim 11 , wherein the microorganism belongs to the bacterial genus Corynebacterium or Brevibacterium.
13. The method of claim 9 , wherein the amino acid is selected from the group consisting of L-lysine, L-threonine and L-methionine.
14. The isolated nucleic acid molecule of claim 1 (a), wherein the amino acid residue at position 75 of SEQ ID NO: 70 is aspartic acid, the amino acid residue at position 332 of SEQ ID NO: 70 is threonine, or the amino acid residue at position 556 of SEQ ID NO: 70 is isoleucine.
15. The isolated nucleic acid molecule of claim 1 (b) wherein nucleotide residues 223-225 of SEQ ID NO: 69 encode aspartic acid, nucleotide residues 994-996 of SEQ ID NO: 69 encode threonine, or nucleotide residues 1666-1668 of SEQ ID NO: 69 encode isoleucine.