Process for the Fermentative Preparation of Organic Chemical Compounds Using Coryneform Bacteria in which the SugR Gene is Present in Attenuated Form

• US 20120214211A1
• Filed: 01/21/2012
• Published: 08/23/2012
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Forschungszentrum Jã¼Lich GmbH (Bundesrepublik Deutschland), Evonik Operations GmbH (Rag-Stiftung)
• Original Assignee: Forschungszentrum Jã¼Lich GmbH (Bundesrepublik Deutschland), Evonik Degussa GmbH
• Inventors: Bathe, Brigitte, Blombach, Bastian, Eikmanns, Bernhard, Engels, Verena, Thierbach, Georg, Wendisch, Volker
• Granted Patent: US 8,592,177 B2
• Time in Patent Office: Days
• US Class Current: 435/115
• CPC Class Codes: C12P 13/06 Alanine; Leucine; Isoleucine; Serine; Homoserine
• CPC Class Codes: C12P 13/08 Lysine; Diaminopimelic acid; Threonine; Valine
• CPC Class Codes: C12P 13/10 Citrulline; Arginine; Ornithine

Abstract

The invention relates to a recombinant coryneform bacterium which secretes an organic chemical compound and in which the sugR gene which codes for a polypeptide having the activity of an SugR regulator has been attenuated. The invention further relates to a processes for using this bacterium for the fermentative preparation of organic chemical compounds.

Claims

1-30. -30. (canceled)

31. A process for the fermentative production of an organic chemical compound, comprising:

a) producing a fermentation broth by fermenting a recombinant coryneform bacterium in a medium comprising a carbon source selected from the group consisting of;

glucose, fructose, sucrose, and acetic acid, wherein said recombinant coryneform bacterium;

i) secretes said organic chemical compound;

ii) has the ability to utilize as a carbon source one or more of the sugars selected from the group consisting of;

glucose, fructose;

sucrose, and acetic acid;

iii) comprises a feedback resistant aspartate kinase; and

iv) comprises a SugR regulator that comprises the sequence of SEQ ID NO;

2 except for one or more differences selected from the group consisting of;

aa) the amino acid at position 37 is selected from the group consisting of;

L-alanine, glycine, L-isoleucine and L-proline;

bb) the amino acid at position 38 is selected from the group consisting of;

L-alanine, glycine, L-isoleucine and L-proline;

cc) the amino acid at position 39 is selected from the group consisting of;

L-alanine, glycine, L-isoleucine and L-proline;

dd) the amino acid at position 40 is L-proline;

ee) the amino acid at position 72 is selected from the group consisting of;

L-alanine, glycine, L-glutamic acid and L-aspartic acid;

ff) the amino acid at position 101 is selected from the group consisting of;

L-arginine, L-lysine, L-phenylalanine, L-methionine, L-glutamine, L-tryptophan, L-tyrosine and L-glutamic acid;

gg) the amino acid at position 105 is selected from the group consisting of;

L-proline, L-phenylalanine, L-isoleucine, L-methionine, L-glutamine, L-tryptophan and L-tyrosine;

hh) the amino acid at position 210 is selected from the group consisting of;

L-alanine, L-arginine, and L-proline; and

ii) the amino acid at position 216 is selected from the group consisting of;

L-alanine, L-glutamic acid, L-isoleucine and L-tryptophan.

b) collecting said fermentation broth containing said organic chemical compound.

32. The process of claim 31, wherein the fermentation broth collected in step b) is dried to form a product based on the fermentation broth.

33. The process of claim 31, wherein the fermentation broth collected in step b) is dried without the removal of biomass.

34. The process of claim 31, wherein biomass is removed from the fermentation broth of step b) so that not more than 30% of the biomass remains in the product thereby produced.

35. The process of claim 31, wherein said organic chemical compound is isolated from the fermentation broth of step b) so that not more than 0.1% of biomass remains in the product thereby produced.

36. The process of claim 31, wherein said organic chemical compound is a hydroxy acid or a keto acid.

37. The process of claim 31, wherein said organic chemical compound is an L-amino acid.

38. The process of claim 31, wherein said organic chemical compound is L-lysine, L-valine or L-isoleucine.

39. The process of claim 31, wherein said recombinant coryneform bacterium is of the species Corynebacterium glutamicum.

40. The process of claim 31, wherein said recombinant coryneform bacterium additionally possesses one or more of the features selected from the group consisting of:

a) overexpression of a polynucleotide which codes for an aspartate-semialdehyde dehydrogenase (Asd),

b) overexpression of a polynucleotide which codes for a dihydrodipicolinate synthase (DapA),

c) overexpression of a polynucleotide which codes for a dihydropicolinate reductase (DapB),

d) overexpression of a polynucleotide which codes for a tetrahydrodipicolinate succinylase (DapD),

e) overexpression of a polynucleotide which codes for a succinyl-aminoketopimelate transaminase (DapC),

f) overexpression of a polynucleotide which codes for a succinyl-diaminopimelate desuccinylase (DapE),

g) overexpression of a polynucleotide which codes for a diaminopimelate dehydrogenase (Ddh),

h) overexpression of a polynucleotide which codes for a diaminopimelate epimerase (DapF),

i) overexpression of a polynucleotide which codes for a diaminopimelate decarboxylase (LysA),

j) overexpression of a polynucleotide which codes for a polypeptide having L-lysine export activity (LysE),

k) overexpression of a polynucleotide which codes for an aspartate aminotransferase (Aat),

l) overexpression of a polynucleotide which codes for a pyruvate carboxylase (Pyc),

m) elimination or attenuation malate-quinone oxidoreductase (Mqo) activity,

n) elimination or attenuation of malate dehydrogenase (Mdh) activity, and

o) elimination or attenuation of citrate synthase (GltA) activity.

41. A process for the fermentative production of an organic chemical compound, comprising:

a) producing a fermentation broth by fermenting a recombinant coryneform bacterium in a medium comprising a carbon source selected from the group consisting of;

glucose, fructose, sucrose, and acetic acid, wherein said recombinant coryneform bacterium;

i) secretes said organic chemical compound;

ii) has the ability to utilize as a carbon source one or more of the sugars selected from the group consisting of;

glucose, fructose;

sucrose, and acetic acid;

iii) comprises a feedback resistant aspartate kinase; and

iv) comprises a gene encoding the SugR regulator of SEQ ID NO;

2, said gene comprising in its promoter region, the nucleotide sequence of SEQ ID NO;

13 except for one or more mutations selected from the group consisting of;

aa) replacement of the nucleobase thymine at position 7 of SEQ ID NO;

13 by guanine;

bb) deletion of one or more of the nucleobases from position 2 to 7 of SEQ ID NO;

13;

cc) deletion of one or more of the nucleobases between position 52 and 57 of SEQ ID NO;

13; and

dd) replacement of one or more of the nucleobases adenine or guanine between position 52 and 57 of SEQ ID NO;

13 by thymine or cytosine;

b) collecting said fermentation broth containing said organic chemical compound.

42. The process of claim 41, wherein the fermentation broth collected in step b) is dried to form a product based on the fermentation broth.

43. The process of claim 41, wherein the fermentation broth collected in step b) is dried without the removal of biomass.

44. The process of claim 41, wherein biomass is removed from the fermentation broth of step b) so that not more than 30% of the biomass remains in the product thereby produced.

45. The process of claim 41, wherein said organic chemical compound is isolated from the fermentation broth of step b) so that not more than 0.1% of biomass remains in the product thereby produced.

46. The process of claim 41, wherein said gene encoding the SugR regulator of SEQ ID NO:

2 comprises a start codon selected from the group consisting of GTG and TTG.

47. The process of claim 41, wherein said organic chemical compound is a hydroxy acid or a keto acid.

48. The process of claim 41, wherein said organic chemical compound is an L-amino acid.

49. The process of claim 41, wherein said organic chemical compound is L-lysine, L-valine or L-isoleucine.

50. The process of claim 41, wherein said recombinant coryneform bacterium is of the species Corynebacterium glutamicum.