Fermentation process for producing itaconic acid under nitrogen free conditions

• US 10,443,077 B2
• Filed: 02/16/2016
• Issued: 10/15/2019
• Est. Priority Date: 02/16/2015
• Status: Active Grant

• Current Assignee: Covestro NV (Covertro Deutschland AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: Weusthuis, Ruud, Vuoristo, Kiira, Eggink, Gerrit, Sanders, Johan Pieter Marinus
• Primary Examiner(s): Fronda, Christian L
• Application Number:
  US15/550,894
• Publication Number:
  US 20180016604A1
• Time in Patent Office: 1,337 Days
• Field of Search: None
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/44 Polycarboxylic acids
• CPC Class Codes: C12Y 401/01006 Aconitate decarboxylase (4.1.1.6)

Abstract

The present invention relates to a method for the production of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid in a suitable fermentation medium, thereby to produce itaconic acid, wherein: (a) the recombinant cell: (i) overexpresses cis-aconitate decarboxylase; and (ii) overexpresses a part of the citric acid cycle and/or has reduced activity of a native metabolic route to acetate and/or lactate; and, optionally, (b) the fermentation is carried out under anaerobic conditions.

Claims

1. A method for the production of itaconic acid, which method comprises fermenting a recombinant cell selected from a bacterial cell or a yeast cell capable of producing itaconic acid in a suitable fermentation medium, thereby to produce itaconic acid, wherein:

(a) the recombinant cell;

(i) overexpresses cis-aconitate decarboxylase; and

(ii) overexpresses a part of the citric acid cycle and/or has reduced activity of a native metabolic route to acetate and/or lactate, wherein overexpression of a part of the citric acid cycle is overexpression of a citrate synthase and/or overexpression of an aconitase and wherein reduced activity of a native metabolic route to acetate is reduced activity of a phosphate acetyltransferase by deleting, disrupting or inactivating the pta gene in the recombinant cell and/or wherein reduced activity of a native metabolic route to lactate is reduced activity of a lactate dehydrogenase by deleting, disrupting or inactivating the ldhA gene in the recombinant cell; and

,

(b) the fermentation is carried out under anaerobic conditions, wherein fermentation carried out under anaerobic conditions means a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed or a fermentation process run under oxygen limited, or micro-aerobical, conditions; and

(c) wherein glutamate production is repressed by carrying out the fermentation under nitrogen free conditions.

2. The method according to claim 1, wherein the cis-aconitate decarboxylase is an Aspergillus terreus cis-aconitate decarboxylase.

3. The method according to claim 1 wherein production of itaconic acid is accompanied by either ethanol and formate production or H₂ and CO₂ production.

4. The method according to claim 1, wherein the bacterial cell is E. coli, or wherein the yeast cell is S. cerevisiae.

5. The method according to claim 1 which comprises recovery of the itaconic acid.

6. A method for the production of itaconic acid, which method comprises fermenting a recombinant bacterial cell capable of producing itaconic acid in a suitable fermentation medium, thereby to produce itaconic acid, wherein:

(a) the recombinant cell;

(i) overexpresses Aspergillus terreus cis-aconitate decarboxylase; and

(ii) overexpresses a part of the citric acid cycle and has reduced activity of a native metabolic route to acetate and lactate, wherein overexpression of a part of the citric acid cycle is overexpression of the gltA gene coding for citrate synthase and overexpression of the acnA gene coding for aconitase and wherein reduced activity of a native metabolic route to acetate is reduced activity of a phosphate acetyltransferase by deleting, disrupting or inactivating the pta gene in the recombinant cell and wherein reduced activity of a native metabolic route to lactate is reduced activity of a lactate dehydrogenase by deleting, disrupting or inactivating the ldhA gene in the recombinant cell; and

,

(b) the fermentation is carried out under anaerobic conditions, wherein fermentation carried out under anaerobic conditions means a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed or a fermentation process run under oxygen limited, or micro-aerobical, conditions; and

,

(c) wherein glutamate production is repressed by carrying out the fermentation under nitrogen free conditions.

7. The method according to claim 6 wherein the recombinant bacterial cell is a recombinant E. coli cell.

8. The method according to claim 6 wherein in step (a) the citrate synthase is C. glutamicum citrate synthase.

9. The method according to claim 6 wherein in step (a) the aconitase is C. glutamicum aconitase.

10. The method according to claim 6 wherein in step (b) the fermentation process is run in the absence of oxygen or in which substantially no oxygen is consumed.