Method for the production of fine chemicals using a
The present invention relates to polynucleotides encoding novel fusion polypeptides essentially composed of a signal peptide for membrane translocation and a polypeptide providing α-1,6-glucosidase activity and to bacteria containing said polynucleotides. The invention further relates to methods for producing fine chemicals using media containing isomaltose and/or panose as carbon source.
1. An isolated polynucleotide encoding a fusion polypeptide comprising the amino acid sequences a), b) and c) wherein:
a) is an N-terminal Tat-signal peptide comprising the amino acid sequence of positions 1 to 33 of SEQ ID NO:10 or positions 1 to 33 of SEQ ID NO:12 or
comprising the amino acid sequence of positions 1 to 33 of SEQ ID NO:10 with Ala at position 13 or positions 1 to 33 of SEQ ID NO:12 with Ala at position 13;
b) is a C-terminal polypeptide having α-1,6-glucosidase activity comprising an amino acid sequence selected from the group consisting of:
b1) a sequence ≥95% identical to the sequence from positions 37 to 639 of SEQ ID NO:10; and
b2) at least (≥) 95% identical to the sequence from positions 37 to 643 of SEQ ID NO:12, and
c) is 0 to 10 amino acid residues between a) and b).
2. The isolated polynucleotide of claim 1 , wherein:
a) the amino acid sequence of b1) is selected from: positions 37 to 639 of SEQ ID NO:10; and from positions 37 to 639 of SEQ ID NO:10 plus an additional Met at the N-terminus in front of position 37 as shown in SEQ ID NO:6; and
b) the sequence of b2) is selected from: positions 39 to 643 of SEQ ID NO:12; positions 38 to 643 of SEQ ID NO:12; and positions 37 to 643 of SEQ ID NO:12.
3. The isolated polynucleotide of claim 1 , wherein the number of amino acid residues of c) is 1 to 3.
4. The isolated polynucleotide of claim 3 , wherein the number of amino acid residues of c) is 3.
5. The isolated polynucleotide of claim 4 , wherein the amino acid residues between a) and b) consist of the amino acid sequence Met Thr Ser.
6. The isolated polynucleotide of claim 4 , wherein the amino acid residues of c) consist of the amino acid sequence Ile Leu Val.
7. The isolated polynucleotide of claim 1 , wherein said polynucleotide is functionally linked to a promotor.
8. The isolated polynucleotide of claim 7 , wherein said promotor is the PtacI promotor comprising the nucleotide sequence of SEQ ID NO:14 from positions 1 to 75 or the PdapBN1 promotor comprising the nucleotide sequence of SEQ ID NO:15.
9. The isolated polynucleotide of claim 8 , wherein said promotor is the PdapBN1-promotor comprising the sequence of SEQ ID NO:15.
10. A bacterium selected from the genus Corynebacterium and comprising the isolated polynucleotide of claim 1 , wherein said bacterium has the ability to secrete a polypeptide having α-1,6-glucosidase activity encoded by said isolated polynucleotide.
11. The bacterium of claim 10 , wherein said isolated polynucleotide is contained in a plasmid vector autonomously replicating in said bacterium or in the chromosome of said bacterium.
12. The bacterium of claim 10 , wherein said bacterium is a Corynebacterium glutamicum.
13. The bacterium of claim 10 , wherein said bacterium has the ability to excrete and produce a fine chemical selected from the group consisting of: L-amino acids; vitamins; nucleosides; and nucleotides.
14. The bacterium of claim 13 , wherein said fine chemical is an L-amino acid.
15. The bacterium of claim 14 , wherein said L-amino acid is selected from the group consisting of: L-lysine; L-threonine; L-valine; and L-isoleucine.
16. A method for producing a fine chemical selected from the group consisting of: L-amino acids; vitamins; nucleosides; and nucleotides; said method comprising culturing the fine chemical producing bacterium according to claim 10 in a suitable medium, where said medium comprises oligomers of a-D-glucose consisting of least two alpha-1-6-glycosidically linked glucose monomers as carbon source.
17. The method of claim 16 , wherein said fine chemical is an L-amino acid.
18. The method of claim 17 , wherein said L-amino acid is selected from the group consisting of: L-lysine; L-threonine; L-valine; and L-isoleucine.