Process for the fermentative preparation of L-ornithine
View Patent ↗A process for the fermentative preparation of L-ornithine using microorganisms characterized by an increased export of the amino acid.
1. A process for preparing L-ornithine, comprising:
a) cultivating in a medium an L-ornithine excreting bacterium selected from the group consisting of Corynebacterium, Bacillus, Streptomyces, Arthrobacter and the Enterobacteriaceae which overexpresses a polynucleotide coding for a polypeptide which has the activity of an L-ornithine exporter and whose amino acid sequence is identical to the amino acid sequence of SEQ ID No. 2 or comprises SEQ ID NO: 2 with a maximum of 25 deletions, insertions, substitutions or N-terminal and/or C-terminal additions of amino acids overall thus producing a fermentation broth, and
b) recovering L-ornithine from the fermentation broth,
wherein plasmid pEC7lysE, deposited as DSM23239, is excluded for overexpression.
2. The process according to claim 1 , wherein the encoded polypeptide comprises the amino acid sequence of SEQ ID No. 2.
3. The process according to claim 1 , wherein said bacterium is Corynebacterium glutamicum , and wherein overexpression increases the level of L-ornithine export activity by at least 10%, compared to the bacteria deposited as ATCC13032, ATCC14067 or ATCC13869.
4. The process according to claim 1 , wherein overexpression is achieved by one or more of the measures selected from the group consisting of
a) increasing the copy number,
b) using a strong promoter, and
c) mutating the promoter, and
d) overexpressing an activator protein.
5. The process according to claim 1 , wherein the bacterium is a Corynebacterium.
6. The process according to claim 1 , wherein the bacterium is Corynebacterium glutamicum.
7. The process according to claim 1 , wherein additionally one or more of the genes selected from the group consisting of
a) odhA gene coding for the E1 subunit of alpha-ketoglutarate dehydrogenase (EC 1.2.4.2),
b) sucA gene coding for dihydrolipoamide succinyl transferase (EC 2.3.1.61),
c) dapA gene coding for a dihydrodipicolinate synthase (DapA, EC 4.2.1.52),
d) dapB gene coding for a dihydrodipicolinate synthase (DapB, EC 1.3.1.26),
e) ddh gene coding for a meso-diaminopimelate dehydrogenase (Ddh, EC 1.4.1.16),
f) lysA gene coding for a diaminopimelate decarboxylase (LysA, EC 4.1.1.20),
g) argR gene coding for a/the repressor (ArgR) of L-arginine biosynthesis,
h) argF gene coding for an ornithine carbamoyl transferase (ArgF, EC 2.1.3.3),
i) argG gene coding for an argininosuccinate synthase (ArgG, EC 6.3.4.5),
j) argH gene coding for an argininosuccinate lyase (ASAL) (ArgH, EC 4.3.2.1),
k) lysC gene coding for an aspartate kinase (LysC, EC 2.7.2.4), and
l) asd gene coding for an aspartate semialdehyde dehydrogenase (Asd, EC 1.2.1.11),
is/are attenuated.
8. The process according to claim 1 , wherein additionally one or more of the genes selected from the group consisting of
a) glutamate dehydrogenase (EC 1.4.1.3) encoded by the gdh gene,
b) glutamate N-acetyltransferase (EC 2.3.1.35 and EC 2.3.1.1) encoded by the argJ gene,
c) acetylglutamatekinase (EC 2.7.2.8) encoded by the argB gene,
d) N-acetyl-gamma-glutamyl-phosphate reductase (EC 1.2.1.38) encoded by the argC gene,
e) acetylornithine aminotransferase (EC 2.6.1.11), encoded by the argD gene,
f) glucose-specific component EIIB (PtsG) (EC 2.7.1.69) of the glucose uptake system, encoded by the ptsG gene,
g) sucrose-specific component EIIB (PtsS) (EC 2.7.1.69) of the sucrose uptake system, encoded by the ptsS gene,
h) glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49) encoded by the zwf gene,
i) glucose-6-phosphate isomerase (EC 5.3.1.9) encoded by the pgi gene,
j) phosphofructokinase (EC 2.7.1.11) encoded by the pfkA gene,
k) fructose-bisphosphate aldolase (EC 4.1.2.13) encoded by the fda gene,
l) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.59) encoded by the gap gene,
m) phosphoglycerate kinase (EC 2.7.2.3) encoded by the pgk gene,
n) pyruvate kinase (EC 2.7.1.40) encoded by the pyk gene,
o) E1 subunit of pyruvate dehydrogenase (EC 1.2.4.1), encoded by the aceE gene,
p) phosphoenolpyruvate carboxylase (EC 4.1.1.31) encoded by the ppc gene,
q) pyruvate carboxylase (EC 6.4.1.1), encoded by the pyc gene,
r) aconitase (EC 4.2.1.3) encoded by the acn gene, and
s) isocitrate dehydrogenase (EC 1.1.1.42) encoded by the icd gene,
is/are enhanced.
9. The process according to claim 1 , which is a process selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process, and continuous process.
10. The process according to claim 1 , wherein L-ornithine or a liquid or solid L-ornithine-containing product is recovered from the L-ornithine-containing fermentation broth.
11. The process according to claim 1 , wherein overexpression is achieved by increasing the copy number.
12. The process according to claim 1 , wherein overexpression is achieved by using a strong promoter.
13. The process according to claim 1 , wherein overexpression is achieved by mutating the promoter.
14. The process according to claim 1 , wherein overexpression is achieved by overexpressing an activator protein.