US2006121581A1PendingUtilityA1

Production of bacterial strains cross reference to related applications

58
Individually held — no corporate assignee on recordPriority: Oct 4, 2002Filed: Oct 3, 2003Published: Jun 8, 2006
Est. expiryOct 4, 2022(expired)· nominal 20-yr term from priority
Inventors:Marguerite A. CervinPhilippe SoucailleDonald E. TrimburFernando Valle
C12N 15/70C12P 7/40C12P 7/46C07K 14/24Y02E50/10C12P 7/44C12P 7/065C12P 7/06C12P 13/22C12P 13/04C12P 7/18C12P 7/28C12P 7/20
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Claims

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

exact text as granted — not AI-modified
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.  
   
   
       33 . A genetically engineered bacterial strain comprising an inactivated endogenous rpoS gene.  
   
   
       34 . The genetically engineered bacterial strain of  claim 33 , wherein the endogenous rpoS gene is deleted.  
   
   
       35 . The genetically engineered bacterial strain of  claim 33  further comprising an overexpressed polypeptide having PEP carboxylase activity.  
   
   
       36 . The genetically engineered bacterial strain of  claim 33 , wherein said bacterial strain has a PTS − /Glu +  phenotype, which was derived from a bacterial strain originally capable of utilizing a PTS for carbohydrate transport.  
   
   
       37 . The genetically engineered bacterial strain of  claim 36 , wherein said bacterial strain is  E. coli.    
   
   
       38 . A method of enhancing the production of an aromatic amino acid in an  E. coli  host cell comprising, a) modifying an  E. coli  host cell by inactivating an endogenous arcA gene and b) culturing the modified host cell in suitable culture media comprising glucose under aerobic conditions to allow production of an aromatic amino acid.  
   
   
       39 . The method according to  claim 38 , wherein the bacterial host cell is a PTS − /Glu +  cell.  
   
   
       40 . The method according to  claim 38  further comprising isolating the aromatic amino acid from the culture media.  
   
   
       41 . The modified  E. coli  host cell obtained according to the method of  claim 38 .  
   
   
       42 . The method according to  claim 38 , wherein the endogenous arcA gene is inactivated by a deletion.

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