Method of constructing amino acid producing bacterial strains, and method of preparing amino acids by fermentation with the constructed amino acid producing bacterial strains

• US 6,962,805 B2
• Filed: 05/25/2000
• Issued: 11/08/2005
• Est. Priority Date: 09/25/1998
• Status: Expired due to Term

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Kimura, Eiichiro, Nakamura, Jun, Ito, Hisao, Matsui, Kazuhiko, Kurahashi, Osamu, Nakamatsu, Tsuyoshi, Asakura, Yoko, Suga, Mikiko, Ohsumi, Tsuyoshi, Kanno, Sohei
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Ramirez, Delia M.
• Application Number:
  US09/577,005
• Publication Number:
  US 20040002143A1
• Time in Patent Office: 1,993 Days
• Field of Search: 536/23.1, 536/23.2, 536/24.1, 435/26, 435/69.1, 435/190, 435/110, 435/471, 435/252.32, 435/189, 435/232, 435/193
• US Class Current: 435/110
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12P 13/14 Glutamic acid; Glutamine

Abstract

A method of producing coryneform bacteria having improved amino acid or nucleic acid productivity comprising the steps of introducing a mutation in a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on a chromosome of a coryneform bacterium to make it close to a consensus sequence, or introducing a change in a promoter sequence of amino acid- or nucleic acid-biosynthesizing genes on a chromosome of a coryneform bacterium by gene recombination to make it close to a consensus sequence, to obtain mutants of the coryneform amino acid- or nucleic acid-producing microorganism, culturing the mutants and selecting a mutant capable of producing the intended amino acid or nucleic acid in a large amount. This method allows the construction of a mutant capable of enriching or controlling the expression of an intended gene without using a plasmid and to promote production of amino acids in a high yield by recombination or mutation.

Claims

1. A method of producing L-glutamic acid, comprising:

culturing a coryneform bacterium expressing an enzyme encoded by a coryneform bacteria glutamic acid biosynthesizing gene, in a medium for a time and under conditions suitable to produce and accumulate said L-glutamic acid in the medium, and

collecting said L-glutamic acid from the medium;

wherein the glutamic acid biosynthesizing gene is located on a chromosome of the coryneform bacterium and the enzyme is selected from the group consisting of glutamate dehydrogenase, citrate synthase, isocitrate dehydrogenase, pyruvate dehydrogenase, and aconitase, said glutamic acid biosynthesizing gene comprising a DNA sequence situated at about position −

35 from the transcription start site of the glutamic acid biosynthesizing gene, wherein said DNA sequence is selected from the group consisting of TTGTCA and TTGCCA,

wherein said producing L-glutamic acid is at a level greater than the production of L-glutamic acid by the corresponding wild-type coryneform bacterium.

2. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes glutamate dehydrogenase.

3. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes citrate synthase.

4. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes isocitrate dehydrogenase.

5. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes pyruvate dehydrogenase.

6. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes aconitase.

7. The method of claim 1, wherein said DNA sequence situated at about position −

35 from the transcription start site of the glutamic acid biosynthesizing gene further comprises TATAAT situated at about position −

10 from the transcription start site.

8. The method of claim 1, wherein said DNA sequence is TTGTCA.

9. The method of claim 8, wherein said glutamic acid biosynthesizing gene further comprises TATAAT situated at about position −

10 from the transcription start site of the gene.

10. The method of claim 8, wherein said glutamic acid biosynthesizing gene further comprises TATAAC situated at about position −

10 from the transcription start site of the gene.

11. The method of claim 1, wherein said DNA sequence is TTGCCA.

12. The method of claim 11, wherein said glutamic acid biosynthesizing gene further comprises TATAAT situated at about position −

10 from the transcription start site of the gene.

13. The method of claim 11, wherein said glutamic acid biosynthesizing gene further comprises TATAAC situated at about position −

10 from the transcription start site of the gene.

14. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes isocitrate dehydrogenase and wherein said DNA sequence is TTGCCA.

15. The method of claim 14, wherein said glutamic acid biosynthesizing gene further comprises TATAAT situated at about position −

10 from the transcription site of the gene.

16. The method of claim 1, wherein said glutamic acid biosynthesizing gene encodes pyruvate dehydrogenase and wherein said DNA sequence is TTGCCA.

17. The method of claim 16, wherein said glutamic acid biosynthesizing gene further comprises TATAAT situated at about position −

10 from the transcription site of the gene.

18. A method of producing L-glutamic acid, comprising:

culturing a coryneform bacterium expressing an enzyme encoded by a coryneform bacteria glutamic acid biosynthesizing gene, in a medium for a time and under conditions suitable to produce and accumulate said L-glutamic acid in the medium, and

collecting said L-glutamic acid from the medium;

wherein the glutamic acid biosynthesizing gene is located on a chromosome of the coryneform bacterium and the enzyme is selected from the group consisting of glutamate dehydrogenase, citrate synthase, isocitrate dehydrogenase, pyruvate dehydrogenase, and aconitase, said glutamic acid biosynthesizing gene comprising a DNA sequence situated at about position −

10 from the transcription start site of the glutamic acid biosynthesizing gene, wherein said DNA sequence is TATAAC,

wherein said producing L-glutamic acid is at a level greater than the production of L-glutamic acid by the corresponding wild-type coryneform bacterium.

19. The method of claim 18, wherein said glutamic acid biosynthesizing gene encodes glutamate dehydrogenase.

20. The method of claim 18, wherein said glutamic acid biosynthesizing gene encodes citrate synthase.

21. The method of claim 20, wherein said glutamic acid biosynthesizing gene further comprises TTGACA situated at about position −

35 from the transcription start site of the gene.

22. The method of claim 18, wherein said glutamic acid biosynthesizing gene encodes isocitrate dehydrogenase.

23. The method of claim 22, wherein said glutamic acid biosynthesizing gene further comprises TTGACA situated at about position −

35 from the transcription start site of the gene.

24. The method of claim 18, wherein said glutamic acid biosynthesizing gene encodes pyruvate dehydrogenase.

25. The method of claim 18, wherein said glutamic acid biosynthesizing gene encodes aconitase.

26. The method of claim 18, wherein said glutamic acid biosynthesizing gene further comprises TTGACA situated at about position −

35 from the transcription site of the gene.