Variants of the promoter of the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase
View Patent ↗The invention relates to an isolated polynucleotide having promoter activity, a variant of the promoter of the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase; and to a microorganism which produces and/or secretes a fine chemical, the microorganism including the isolated polynucleotide having promoter activity, which enables various genes to be overexpressed in comparison with the particular starting strain; and to a process for preparing fine chemicals using the microorganism.
1. A process for preparing a fine chemical, comprising
a) fermenting a microorganism in a fermentation medium to form a fermentation broth, wherein the microorganism comprises an isolated polynucleotide having promoter activity, which comprises SEQ ID NO:3 or SEQ ID NO:34
b) accumulating the fine chemical in the fermentation broth of a) and/or in the cells of the microorganism.
2. The process for preparing a fine chemical according to claim 1 , which is a batch process, a fed-batch process, a repeated fed-batch process, or a continuous process.
3. The process according to claim 1 , further comprising recovering the fine chemical or a liquid or solid fine chemical-containing product from the fine chemical-containing fermentation broth.
4. The process according to claim 1 , wherein the isolated polynucleotide is contained in a vector.
5. The process according to claim 1 , wherein the isolated polynucleotide is integrated in a chromosome of the microorganism.
6. The process according to claim 1 , wherein the microorganism is a Corynebacterium.
7. The process according to claim 1 , wherein the fine chemical is an L-amino acid or an organic acid.
8. The process according to claim 1 , wherein the fine chemical is an L-amino acid selected from the group consisting of L-isoleucine, L-lysine, L-methionine, L-ornithine, L-proline, L-valine, and L-tryptophan.
9. The process according to claim 1 , wherein the fine chemical is an organic acid is a ct-keto acid selected from the group consisting of a-ketoisocaproic acid, a-ketovaleric acid, and a-keto-[3-methylvaleric acid.
10. The process according to claim 4 , wherein the polynucleotide is functionally linked at the 3′ end to a second polynucleotide which comprises an ATG or GTG start codon at the 5′ end and the second polynucleotide codes for one or more polypeptides, wherein said link is a direct link, a link through a linker oligonucleotide, or a link through a linker polynucleotide.
11. The process according to claim 4 , wherein the polynucleotide of SEQ ID NO:3 or SEQ ID NO:34 is linked through the adenosine nucleotide in position 461 at the 3″ end of SEQ ID NO:3 or SEQ ID NO:34 by a linker oligonucleotide of 1, 2, 3, 4 or 5 nucleotides in length to the first nucleotide of the start codon of the second polynucleotide.
12. The process according to claim 4 , wherein the polynucleotide of SEQ ID NO:3 or SEQ ID NO:34 is linked through the adenosine nucleotide at its 3′ end, by a linker polynucleotide of from 6 to 600 nucleotide in length to the first nucleotide of the start codon of the second polynucleotide.
13. The process according to claim 12 , wherein the linker polynucleotide comprises a nucleotide sequence which ensures translation of RNA transcribed from the polynucleotide.
14. The process according to claim 12 , wherein the sequence of the linker polynucleotide is SEQ ID NO: 12 or SEQ ID NO: 13.
15. The process according to claim 12 , wherein the sequence of the linker polynucleotide is SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.
16. The process according to claim 10 , wherein the isolated polynucleotide is functionally linked through the adenosine nucleotide in position 461 at the 3′ end of SEQ ID NO:3 or SEQ ID NO:34 to a linker oligonucleotide or to a linker polynucleotide which ensures translation of RNA.
17. The process according to claim 10 , wherein the second polynucleotide which codes for one or more polypeptides is selected from the group consisting of:
a) Polynucleotide coding for the Zwf subunit of glucose-6-phosphate 1-dehydrogenase (Zwf),
b) Polynucleotide coding for the OpcA subunit of glucose-6-phosphate 1-dehydrogenase (OpcA),
c) Polynucleotide coding for a 6-phosphogluconolactonase (DevB, EC NO.: 3.1.1.31),
d) Polynucleotide coding for a transketolase (Tkt),
e) Polynucleotide coding for a transaldolase (Tal),
f) Polynucleotide coding for a 6-phosphogluconate dehydrogenase (Gnd),
g) Polynucleotide coding for a ribulose-phosphate 3-epimerase (Rpe),
h) Polynucleotide coding for a ribose-5-phosphate isomerase B (Rpi),
i) Polynucleotide coding for a phosphoenolpyruvate carboxylase (Ppc),
j) Polynucleotide coding for a fructose 1,6-bisphosphatase (Fbp),
k) Polynucleotide coding for a pyruvate carboxylase (Pyc),
l) Polynucleotide coding for a dihydrodipicolinate synthase (DapA),
m) Polynucleotide coding for an aspartate-semialdehyde dehydrogenase (Asd),
n) Polynucleotide coding for a meso-diaminopimelate dehydrogenase (Ddh),
o) Polynucleotide coding for a diaminopimelate decarboxylase (LysA),
p) Polynucleotide coding for an aspartate aminotransferase (Aat),
q) Polynucleotide coding for a polypeptide having L-lysine-export activity (LysE),
r) Polynucleotide coding for a dihydrodipicolinate reductase (DapB),
s) Polynucleotide coding for an aspartate kinase (LysC),
t) Polynucleotide coding for a succinyldiaminopimelate aminotransferase, AT class I (DapC),
u) Polynucleotide coding for a tetrahydrodipicolinate succinylase (DapD),
v) Polynucleotide coding for a succinyl-diaminopimelate desuccinylase (DapE),
w) Polynucleotide coding for a diaminopimelate epimerase (DapF),
x) Polynucleotides coding for the subunits of an acetolactal synthase (IlvBN),
y) Polynucleotide coding for an isomeroreductase (IlvC),
z) Polynucleotide coding for a dihydroxy-acid dehydratase (IlvD),
aa) Polynucleotide coding for a transaminase (IlvE),
bb) Polynucleotide coding for a threonine dehydratase (IlvA),
cc) Polynucleotide coding for a homoserine dehydrogenase (Hom),
dd) Polynucleotide coding for a homoserine kinase (ThrB),
ee) Polynucleotide coding for a threonine synthase (ThrC),
if) Polynucleotide coding for an isopropylmalate synthase (LeuA),
gg) Polynucleotide coding for an isopropylmalate dehydrogenase (LeuB),
hh) Polynucleotide coding for the large subunit of an isopropylmalate isomerase (LeuC),
ii) Polynucleotide coding for the small subunit of an isopropylmalate isomerase (LeuD),
jj) Polynucleotide coding for a lysine/omithine transporter,
kk) Polynucleotide coding for an N-acetylglutamate kinase (ArgB),
ll) Polynucleotide coding for a glutamate dehydrogenase (Gdh),
mm) Polynucleotide coding for a glutamate N-acetyltransferase (ArgJ),
nn) Polynucleotide coding for an N-acetyl-gamma-glutamyl-phosphate reductase (ArgC), and
oo) Polynucleotide coding for an acetylomithine aminotransferase (ArgD).