IP Library › Granted Patent US 9,045,762

Granted Patent B2

US 9,045,762 · App. 14/532,259 · Granted Jun 2, 2015

Variants of the promoter of the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase

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Quick Facts

Patent No.: US 9,045,762
App. No.: 14/532,259
Granted: Jun 2, 2015
Kind: B2

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USPTO Patent Center↗ Full Patent PDF↗

Abstract

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.

Claims (102)

1. A vector comprising an isolated polynucleotide having promoter activity, which comprises SEQ ID NO:3 or SEQ ID NO:34.

2. The vector according to claim 1 , 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.

3. The vector according to claim 2 , 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.

4. The vector according to claim 2 , 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.

5. The vector according to claim 4 , wherein the linker polynucleotide comprises a nucleotide sequence which ensures translation of RNA transcribed from the polynucleotide.

6. The vector according to claim 4 , wherein the sequence of the linker polynucleotide is SEQ ID NO:12 or SEQ ID NO:13.

7. The vector according to claim 4 , 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.

8. The vector according to claim 1 , 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.

9. The vector according to claim 2 , wherein the second polynucleotide which codes for one or more polypeptides is selected from the group consisting of:

a) Polynucleotide (zwf gene) coding for the Zwf subunit of glucose-6-phosphate 1-dehydrogenase (Zwf),

b) Polynucleotide (opcA gene) coding for the OpcA subunit of glucose-6-phosphate 1-dehydrogenase (OpcA),

c) Polynucleotide (devB gene) coding for a 6-phosphogluconolactonase (DevB),

d) Polynucleotide (tkt gene) coding for a transketolase (Tkt),

e) Polynucleotide (tal gene) coding for a transaldolase (Tal),

f) Polynucleotide (gnd gene) coding for a 6-phosphogluconate dehydrogenase (Gnd),

g) Polynucleotide (rpe gene) coding for a ribulose-phosphate 3-epimerase (Rpe),

h) Polynucleotide (rpi gene) coding for a ribose-5-phosphate isomerase B (Rpi),

i) Polynucleotide (ppc gene) coding for a phosphoenolpyruvate carboxylase (Ppc),

j) Polynucleotide (fbp gene) coding for a fructose 1,6-bisphosphatase (Fbp),

k) Polynucleotide (pyc gene) coding for a pyruvate carboxylase (Pyc),

l) Polynucleotide (dapA gene) coding for a dihydrodipicolinate synthase (DapA),

m) Polynucleotide (asd gene) coding for an aspartate-semialdehyde dehydrogenase (Asd),

n) Polynucleotide (ddh gene) coding for a meso-diaminopimelate dehydrogenase (Ddh),

o) Polynucleotide (lysA gene) coding for a diaminopimelate decarboxylase (LysA),

p) Polynucleotide (aat gene) coding for an aspartate aminotransferase (Aat),

q) Polynucleotide (lysE gene) coding for a polypeptide having L-lysine-export activity (LysE),

r) Polynucleotide (dapB gene) coding for a dihydrodipicolinate reductase (DapB),

s) Polynucleotide (lysC gene) coding for an aspartate kinase (LysC),

t) Polynucleotide (dapC gene) coding for a succinyldiaminopimelate aminotransferase, AT class I (DapC),

u) Polynucleotide (dapD gene) coding for a tetrahydrodipicolinate succinylase (DapD),

v) Polynucleotide (dapE gene) coding for a succinyl-diaminopimelate desuccinylase (DapE),

w) Polynucleotide (dapF gene) coding for a diaminopimelate epimerase (DapF),

x) Polynucleotides (ilvB gene and ilvN gene) coding for the subunits of an acetolactate synthase (IlvBN),

y) Polynucleotide (ilvC gene) coding for an isomeroreductase (IlvC),

z) Polynucleotide (ilvD gene) coding for a dihydroxy-acid dehydratase (IlvD),

aa) Polynucleotide (ilvE gene) coding for a transaminase (IlvE),

bb) Polynucleotide (ilvA gene) coding for a threonine dehydratase (IlvA),

cc) Polynucleotide (hom gene) coding for a homoserine dehydrogenase (Hom),

dd) Polynucleotide (thrB gene) coding for a homoserine kinase (ThrB),

ee) Polynucleotide (thrC gene) coding for a threonine synthase (ThrC),

ff) Polynucleotide (leuA gene) coding for an isopropylmalate synthase (LeuA),

gg) Polynucleotide (leuB gene) coding for an isopropylmalate dehydrogenase (LeuB),

hh) Polynucleotide (leuC gene) coding for the large subunit of an isopropylmalate isomerase (LeuC),

ii) Polynucleotide (leuD gene) coding for the small subunit of an isopropylmalate isomerase (LeuD),

jj) Polynucleotide (lysE gene) coding for a lysine/ornithine transporter,

kk) Polynucleotide (argB gene) coding for an N-acetylglutamate kinase (ArgB),

ll) Polynucleotide (gdh gene) coding for a glutamate dehydrogenase (Gdh),

mm) Polynucleotide (argJ gene) coding for a glutamate N-acetyltransferase (ArgJ),

nn) Polynucleotide (argC gene) coding for an N-acetyl-gamma-glutamyl-phosphate reductase (ArgC), and

oo) Polynucleotide (argD gene) coding for an acetylornithine aminotransferase (ArgD).

10. A microorganism comprising an isolated polynucleotide having promoter activity, which comprises SEQ ID NO:3 or SEQ ID NO:34.

11. A microorganism comprising the vector according to claim 1 .

12. The microorganism according to claim 10 , wherein the polynucleotide is integrated in the chromosome.

13. The microorganism according to claim 10 , which is a bacterium of the genus Corynebacterium.

14. The microorganism according to claim 13 , wherein the isolated polynucleotide having promoter activity in the chromosome

1. has been replaced with a native promoter at a native gene locus of the second polynucleotide, or

2. has been integrated with the second polynucleotide at a native gene locus of the second polynucleotide or at another gene locus, wherein the second polynucleotide which codes for one or more polypeptides is selected from the group consisting of: