Glucose transport mutants for production of biomaterial
AssigneeDanisco US Inc.
InventorsMarguerite A. Cervin, Philippe Soucaille, Fernando Valle, Gregory M. Whited
Life of this patent
Estimating the term…
Abstract
A method is disclosed for restoring a Glu+ phenotype to a PTS−/Glu− bacterial cell which was originally capable of utilizing a phosphotransferase transport system (PTS) for carbohydrate transport. Bacterial cells comprising the Glu+ phenotype have modified endogenous chromosomal regulatory regions which are operably linked to polynucleotides encoding galactose permeases and glucokinases.
Claims20
A method for increasing the production of a desired product in a PTS−/Glu− bacterial host cell originally capable of utilizing a PTS for carbohydrate transport comprising,
- a) transforming a bacterial host cell having a PTS−/Glu− phenotype with a DNA construct comprising a promoter, wherein said DNA construct is chromosomally integrated into the PTS−/Glu− host cell replacing an endogenous promoter which is operably linked to a nucleic acid encoding a glucose assimilation protein;
- b) culturing the transformed bacterial host cell under suitable conditions;
- c) allowing expression of the glucose assimilation protein to obtain a host cell having a PTS−/Glu+ phenotype;
- and
- d) obtaining an increased amount of a desired product in the transformed bacterial host cell compared to the amount of the desired product produced in a corresponding PTS bacterial cell cultured under essentially the same culture conditions,
- wherein said desired product is selected from the group consisting of pyruvate, PEP, lactate, acetate, glycerol, ethanol, succinate and chorismate.
A method of restoring a Glu+ phenotype to a PTS−/Glu− bacterial host cell which was originally capable of utilizing a phosphotransferase transport system (PTS) for carbohydrate transport comprising
- a) modifying an endogenous chromosomal regulatory region which is operably linked to a nucleic acid encoding a glucose transporter in a PTS−/Glu− host cell by transforming the PTS−/Glu− host cell with a first DNA construct comprising a promoter and DNA flanking sequences corresponding to upstream (5′) regions of the glucose transporter;
- b) allowing integration of the first DNA construct, wherein the first DNA construct replaces an endogenous promoter of the nucleic acid encoding the glucose transporter;
- and
- c) allowing expression of the glucose transporter, wherein said expression restores a Glu+ phenotype to the PTS−/Glu− host cell.
A method of increasing phosphoenolpyruvate (PEP) availability in a bacterial host cell comprising,
- a) selecting a bacterial host cell having a PTS−/Glu− phenotype, wherein the bacterial host was originally capable of utilizing a phosphotransferase transport system (PTS) for carbohydrate transport;
- b) modifying an endogenous chromosomal regulatory sequence of the selected bacterial host cell comprising transforming said selected bacterial host cell with a DNA construct comprising a promoter, wherein said DNA construct is chromosomally integrated into the selected bacterial host cell replacing an endogenous promoter which is operably linked to a nucleic acid encoding a glucose assimilation protein;
- c) culturing the transformed bacterial host cell under suitable conditions;
- and
- d) allowing expression of the glucose assimilation protein to obtain an altered host cell having a PTS−/Glu+ phenotype, wherein the PEP availability is increased compared to the PEP availability in a corresponding unaltered PTS bacterial host cell cultured under essentially the same culture conditions.
Dependencies are inferred from the claim text.