Production of Bacterial Strains Cross Reference To Related Applications

• US 20090075347A1
• Filed: 07/11/2008
• Published: 03/19/2009
• Est. Priority Date: 10/04/2002
• Status: Active Grant

• Current Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Inventors: Valle, Fernando, Cervin, Marguerite A., Soucaille, Philippe, Trimbur, Donald E.
• Granted Patent: US 8,852,890 B2
• Time in Patent Office: Days
• US Class Current: 435/106
• CPC Class Codes: C07K 14/24 from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12P 13/04 Alpha- or beta- amino acids other amino acids C12P13/005
• CPC Class Codes: C12P 13/22 Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/065 with microorganisms other than yeasts
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/20 Glycerol
• CPC Class Codes: C12P 7/28 Acetone-containing products
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/44 Polycarboxylic acids
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention provides methods to enhance production of desired products and increase the growth rate of a bacterial strain by inactivating an endogenous arcA and optionally overexpressing a ppc gene.

Claims

1. A method of enhancing the production of a desired product in a bacterial host cell comprising, a) modifying a bacterial host cell by inactivating an endogenous arcA gene and b) culturing the modified bacterial host cell in suitable culture media comprising glucose under aerobic conditions to allow production of a desired product.

2. The method according to claim 1, wherein the bacterial host cell is from a strain of the Enterobacteriaceae family.

3. The method according to claim 2, wherein the bacterial host cell is an E. coli or Pantoea cell.

4. The method according to claim 1, wherein the bacterial host cell is a PTS⁻

/Glu+ cell.

5. The method according to claim 1, wherein the desired product is selected from the group consisting of glycerol, PEP, pyruvate, chorismate, ethanol, succinate and dihydroxyacetone-P.

6. The method according to claim 5, wherein the desired product is chorismate.

7. The method according to claim 6, wherein the chorismate is further converted to an aromatic amino acid.

8. The method according to claim 1 further comprising inactivating the expression of an endogenous gene encoding a polypeptide having RpoS activity, Edd activity, Pta activity, AckA activity or MgsA activity.

9. The method according to claim 1 further comprising transforming the bacterial host cell with a DNA fragment comprising an exogenous promoter, wherein the DNA fragment including the exogenous promoter is integrated into the host cell chromosome and replaces the endogenous promoter which is operably linked to a PEP carboxylase coding sequence wherein PEP carboxylase is overexpressed.

10. The method according to claim 1 further comprising isolating the desired product from the culture media.

11. The modified bacterial host cell obtained according to the method of claim 1.

12. A method of enhancing biomass production in bacterial host cells comprising a) modifying a bacterial host cell by inactivating an endogenous arcA gene and b) culturing the modified bacterial cell under suitable culture conditions wherein said culture conditions include aerobic fermentation and glucose as a carbon source and wherein biomass production is enhanced in the modified bacterial cell compared to biomass production in a corresponding non-modified bacterial cell cultured under essentially the same conditions.

13. The method according to claim 12 further comprising inactivating an endogenous rpoS gene.

14. The method according to claim 12, wherein the endogenous arcA gene is inactivated by a deletion.

15. The method according to claim 12, further comprising inactivating an endogenous gene encoding a polypeptide having phosphogluconate dehydratase activity, phosphotransacetylase activity, acetyl kinase activity or methylglyoxyal synthase activity.

16. The method according to claim 15, wherein the endogenous gene encoding a polypeptide having phosphogluconate dehydratase activity is an edd gene.

17. The method according to claim 15, wherein the endogenous gene encoding a polypeptide having phosphotransacetylase activity is a pta gene.

18. The method according to claim 15, wherein the endogenous gene encoding a polypeptide having acetyl kinase activity is an ackA gene.

19. The method according to claim 15, wherein the endogenous gene encoding a polypeptide having methylglyoxyal synthase activity is a mgsA gene.

20. The method according to claim 12 further comprising isolating the modified bacterial cell.

21. The method according to claim 12, wherein the bacterial host cell is selected from the group consisting Escherichia cells, Pantoea cells, Klebsiella cells, Gluconobacter cells and Erwinia cells.

22. The method according to claim 21, wherein the bacterial host cell is an E. coli cell or a Pantoea cell.

23. The modified bacterial cells obtained according to the method of claim 12.

24. A genetically engineered bacterial strain of the Enterobacteriacea family comprising an inactivated endogenous arcA gene and an overexpressed polypeptide having PEP carboxylase activity.

25. The genetically engineered bacterial strain of claim 24, wherein said strain is selected from the genus consisting of Escherichia, Pantoea, Klebsiella, Gluconobacter and Erwinia,

26. The genetically engineered bacterial strain of claim 24, wherein said strain is a strain of E. coli.

27. The genetically engineered bacterial strain of claim 24, wherein the endogenous arcA gene is deleted.

28. The genetically engineered bacterial strain of claim 24 further comprising an inactivated endogenous mgsA.

29. The genetically engineered bacterial strain of claim 24 further comprising an inactivated endogenous edd.

30. The genetically engineered bacterial strain of claim 24, further comprising an inactivated endogenous rpoS.

31. The genetically engineered bacterial strain of claim 24, wherein the overexpressed polypeptide having PEP carboxylase is operably linked to an exogenous promoter.

32. The genetically engineered bacterial strain of claim 31, wherein the exogenous promoter is a GI promoter.

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