Metabolic flux control at the pyruvate node in an anaerobic Escherichia coli strain with an active pyruvate dehydrogenase.
During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y(ATP)) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y(ATP) suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.
Wang
Qingzhao
Q
Department of Microbiology and Cell Science, Box 110700, University of Florida, Gainesville, FL 32611, USA.
Ou
Mark S
MS
Kim
Y
Y
Ingram
L O
LO
Shanmugam
K T
KT
D000123:Acetyltransferases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000693:Anaerobiosis; D004734:Energy Metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011768:Pyruvate Dehydrogenase Complex; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D000123:Acetyltransferases; C022560:formate C-acetyltransferase