Biochemical synthesis of 1,4-butanediamine

• US 8,497,098 B2
• Filed: 07/11/2005
• Issued: 07/30/2013
• Est. Priority Date: 07/15/2004
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: Eppelmann, Katrin, Nossin, Petrus M. M., Kremer, Susanne M., Wubbolts, Marcel G., Raeven, Leon J. R. M.
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US11/632,458
• Publication Number:
  US 20090011478A1
• Time in Patent Office: 2,941 Days
• Field of Search: None
• US Class Current: 435/128
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 13/001 Amines; Imines

Abstract

The invention relates to a process for biochemical synthesis of 1,4-butanediamine in a microorganism having an increased level of an ornithine decarboxylase activity (increased ODC activity) as compared to the native level of the ornithine decarboxylase activity, wherein in the microorganism also an increased activity of N-acetylglutamate formation is present as compared to the native level of activity of N-acetylglutamate formation in the microorganism and wherein 1,4-butanediamine produced in the microorganism is excreted into a fermentation broth, and is recovered from the fermentation broth. The invention also relates to vectors, plasmids and hosts carrying a corresponding increased ODC activity and an increased activity of N-acetylglutamate formation.

Claims

1. Process for biochemical synthesis of 1,4-butanediamine in a microorganism having an increased level of ornithine decarboxylase (ODC) activity as compared to the native level of ornithine decarboxylase activity, wherein there is increased N-acetylglutamate formation in said microorganism as compared to the native level of N-acetylglutamate formation;

the process comprising;

(a) culturing said microorganism to excrete 1,4-butanediamine into a fermentation broth, whereby said increased ODC activity is achieved by overexpression of an ornithine decarboxylase encoding gene speF or speC, each belonging to E.C.4.1.1.17, and the increased activity of N-acetylglutamate formation is achieved by overexpression of at least one of an N-acetyleglutamate synthase encoding argA (belonging to E.C.2.3.1.1) and an N²-acetyl-L-ornithine;

L-gluamate N-acetyl transferase encoding gene argJ (belonging to E.C.2.3.1.35), and

(b) recovering said 1,4-butanediamine from the fermentation broth.

2. Process according to claim 1, wherein the ornithine decarboxylase encoding gene speF or speC originate from one of the genera selected from the group consisting of Escherichia, Shigella, Salmonella, Yersinia, and Shewanella.

3. Process according to claim 2, wherein the ornithine decarboxylase encoding gene is originating from one of the species selected from the group consisting of Escherichia coli, Shigella flexneri, Salmonella typhimutium, Yersinia pestis, and Shewanella oneidensis.

4. Process according to claim 1, wherein the ornithine decarboxylase encoding gene is speF originating from one of the species selected from the group consisting of Escherichia coli, Salmonella typhimutium, and Shewanella oneidensis.

5. Process according to claim 1, wherein the increased activity of N-acetylglutamate formation is achieved by overexpression of an N-acetylglutamate synthase encoding gene argA originating from one of the genera selected from the group consisting of Escherichia, Shigella, Salmonella, Yersinia, Photorhabdus, and Buchnera.

6. Process according to claim 5, wherein the increased activity of N-acetylglutamate formation is achieved by overexpression of an N-acetylglutamate synthase encoding gene argA originating from one of the species selected from the group consisting of Escherichia coli, Shigella flexneri, Salmonella enterica, Yersinia pestis, Photorhabdus luminescens, and Buchnera aphidicola.

7. Process according to claim 1, wherein the increased activity of N-acetylglutamate formation is achieved by overexpression of an N²-acetyl-L-ornithine:

L-glutamate N-acetyl transferase encoding gene argJ (gene encoding an enzyme classified (E.C.) in group E.G. 2. 3.1.35) originating from one of the genera selected from the group consisting of Bacillus, Listeria, Oceanobacillus, Staphylococcus, Lactobacillus, Corynebacterium, Mycobacterium, Thermobifida, Streptomyces, and Bifidobacterium.

8. Process according to claim 7, wherein the increased activity of N-acetylglutamate formation is achieved by overexpression of an N²-acetyl-L-ornithine:

L-glutamate N-acetyl transferase encoding gene originating from one of the species selected from the group consisting of Bacillus cereus, Listeria monocytogenes, Oceanobacillus iheyensis, Staphylococcus epidermis, Lactobacillus plantarum, Lactobacillus lactis, Corynebacterium glutamicum, Mycobacterium leprae, Thermobifida fusca, Streptomyces coelicor, and Bifidobacterium longum.

9. Process according to claim 1, wherein additionally also an increased enzyme activity is obtained for at least two other enzymes by overexpression of either

(i) an arginine decarboxylase encoding gene speA (gene encoding an enzyme classified (E.C.) in group E.C. 4.1.1.19) and an agmatinase encoding gene speB (belonging to E.C. 3.5.3.11;

also referred to as agmatine ureahydrolase encoding gene);

or

(ii) an arginine decarboxylase encoding gene speA (gene encoding an enzyme classified (E.C.) in group E.C. 4.1.1.19), and an agmatine iminohydrolase encoding gene aguA (belonging to E.C. 3.5.3.12;

also referred to as agmatine deiminase encoding gene), and an N-carbamoylputrescine amidohydrolase encoding gene aguB (gene encoding an enzyme classified (E.C.) in group E.C. 3.5.1.53), and optionally also an agmatinase encoding gene speB (gene encoding an enzyme classified (E.C.) in group E.C. 3.5.3.11).

10. Process according to claim 9, wherein the overexpressed arginine decarboxylase encoding gene is an arginine decarboxylase gene speA originating from one of the genera selected from the group consisting of Escherichia, Shigella, Salmonella, Yersinia, Pasteurella, and Neisseria.

11. Process according to claim 10, wherein the overexpressed arginine decarboxylase encoding gene is an arginine decarboxylase gene speA originating from one of the species selected from the group consisting of Escherichia coli, Shigella flexneri, Salmonella enterica, Yersinia pestis, Pasteurella multocida, and Neisseria meningitidis.

12. Process according to claim 9, wherein the overexpressed agmatinase encoding gene is an agmatinase gene speB originating from one of the genera selected from the group consisting of Escherichia, Salmonella, Proteus, Photorhabdus, Vibrio, and Neisseria.

13. Process according to claim 12, wherein the overexpressed agmatinase encoding gene is an agmatinase gene speB originating from one of the species selected from the group consisting of Escherichia coli, Salmonella enterica, Proteus mirabilis, Photorhabdus luminescens, Vibrio cholerae, and Neisseria meningitidis.

14. Process according to claim 9, wherein the overexpressed agmatine iminohydrolase encoding gene and/or the overexpressed N-carbamoylputrescine amidohydrolase encoding gene is an agmatine iminohydrolase gene aguA and/or an N-carbamoylputrescine amidohydrolase gene aguB originating from one of the genera selected from the group consisting of Pseudomonas, Streptococcus, Streptomyces, Azotobacter, Arabidopsis, Novosphingobium, and Bacillus.

15. Process according to claim 14, wherein the overexpressed agmatine iminohydrolase encoding gene and/or the overexpressed N-carbamoylputrescine amidohydrolase encoding gene is an agmatine iminohydrolase gene aguA and/or an N-carbamoylputrescine amidohydrolase gene aguB originating from one of the species selected from the group consisting of Pseudomonas aeruginosa, Streptococcus mutans, Streptomyces avermitilis, Azotobacter vinelandii, Arabidopsis thaliana, Novosphingobium aromaticivorans, and Bacillus cereus.

16. Process according to claim 1, wherein the process is carried out in a host organism selected from the group consisting of Saccharomyces sp., Bacillus sp., Corynebacterium sp., Escherichia sp., and Pichia sp.

17. Process according to claim 1, wherein the process is carried out in a host organism selected from the group of Saccharomyces cerevisiae, Corynebacterium sp., and Escherichia sp. and in that, apart from the increased level of activity of an ornithine decarboxylase and of N-acetylglutamate formation, at least also the level of activity of an arginine decarboxylase in combination with an agmatinase and/or an agmatine iminohydrolase and an N-carbamoylputrescine amidohydrolase is increased.

18. An isolated microorganism having an increased level of ornithine decarboxylase (ODC) activity as compared to the native level of ornithine decarboxylase activity and an increased activity of N-acetylglutamate formation as compared to the native level of activity of N-acetylglutamate formation, whereby said increased ODC activity is achieved by overexpression of an ornithine decarboxylase encoding gene speF or speC, each belonging to E.C.4.1.1.17, and the increased activity of N-acetylglutamate formation is achieved by overexpression of at least one of an N-acetyleglutamate synthase encoding argA (belonging to E.C.2.3.1.1) and an N²-acetyl-L-ornithine:

L-gluamate N-acetyl transferase encoding gene argJ (belonging to E.C.2.3.1.35).

19. Microorganism according to claim 18, wherein there is an increased level of activity of one or more further enzyme activities selected from the group consisting of arginine decarboxylase, agmatinase, agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase.