Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli.
During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzyme-dominated CO(2) fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.
Zhang
Xueli
X
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA.
Jantama
Kaemwich
K
Moore
Jonathan C
JC
Jarboe
Laura R
LR
Shanmugam
Keelnatham T
KT
Ingram
Lonnie O
LO
D000255:Adenosine Triphosphate / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D019143:Evolution, Molecular; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D007218:Industrial Microbiology / Q000379:methods*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D033902:Periplasmic Binding Proteins / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics; D019802:Succinic Acid / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D002245:Carbon Dioxide; D000255:Adenosine Triphosphate; D019802:Succinic Acid; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D019764:Phosphoenolpyruvate Carboxykinase (ATP)