Process for the fermentative preparation of L-ornithine

• US 8,951,759 B2
• Filed: 02/20/2014
• Issued: 02/10/2015
• Est. Priority Date: 03/30/2010
• Status: Active Grant

• Current Assignee: Evonik Operations GmbH (Rag-Stiftung)
• Original Assignee: Evonik Degussa GmbH
• Inventors: Claes, Wilfried, Gerstmeir, Robert
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US14/185,088
• Publication Number:
  US 20140206068A1
• Time in Patent Office: 355 Days
• Field of Search: 435/114
• US Class Current: 435/114
• CPC Class Codes: C12N 15/77 for Corynebacterium; for Brevibacterium
• CPC Class Codes: C12P 13/10 Citrulline; Arginine; Ornithine

Abstract

A process for the fermentative preparation of L-ornithine using microorganisms characterized by an increased export of the amino acid.

Claims

1. An isolated or purified and genetically modified L-ornithine excreting bacterium selected from the group consisting of Corynebacterium, Bacillus, Streptomyces, Arthrobacter and the Enterobacteriaceae that expresses a polynucleotide encoding a L-ornithine exporter polypeptide 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 overall.

2. The isolated or purified L-ornithine excreting bacterium of claim 1 that has been genetically modified to express the L-ornithine exporter in a greater amount than the corresponding unmodified bacterium.

3. The isolated or purified L-ornithine excreting bacterium of claim 1 that is a Corynebacterium.

4. The isolated or purified L-ornithine excreting bacterium of claim 1 that is Corynebacterium glutamicum.

5. The isolated or purified L-ornithine-excreting bacterium of claim 1, which expresses a polynucleotide encoding a L-ornithine exporter polypeptide whose amino acid sequence is identical to the amino acid sequence of SEQ ID No. 2.

6. The isolated or purified L-ornithine-excreting bacterium of claim 1, which expresses a polynucleotide encoding a L-ornithine exporter polypeptide comprising SEQ ID No. 2 with a maximum of 25 deletions, insertions, substitutions or N-terminal and/or C-terminal additions overall.

7. The isolated or purified L-ornithine-excreting bacterium of claim 1, which is Corynebacterium glutamicum, and wherein expression increases the level of L-ornithine export activity by at least 10%, compared to the bacteria deposited as ATCC13032, ATCC14067 or ATCC13869.

8. The isolated or purified L-ornithine-excreting bacterium of claim 1, wherein expression 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.

9. The isolated or purified L-ornithine-excreting bacterium of claim 1, wherein additionally one or more of the genes selected from the group consisting of a) odhA gene coding for the El 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 1) asd gene coding for an aspartate semialdehyde dehydrogenase (Asd, EC 1.2.1.11), is/are attenuated in said bacterium.

10. The isolated or purified L-ornithine-excreting bacterium of 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, 1) 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) El 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 (EC6.4.1.1), 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.

11. The isolated or purified L-ornithine-excreting bacterium of claim 1, wherein expression is overexpression.

12. The isolated or purified L-ornithine-excreting bacterium of claim 11, wherein overexpression is achieved by increasing the copy number.

13. The isolated or purified L-ornithine-excreting bacterium of claim 11, wherein overexpression is achieved by using a strong promoter.

14. The isolated or purified L-ornithine-excreting bacterium of claim 11, wherein overexpression is achieved by mutating the promoter.

15. The isolated or purified L-ornithine-excreting bacterium of claim 11, wherein overexpression is achieved by overexpressing an activator protein.