The Glycolytic Flux in Escherichia coli Is Controlled by the Demand for ATP

Abstract

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F 1 part of the membrane-bound (F 1 F 0 ) H ؉ -ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.The glycolytic pathway of various organisms has been exploited for thousands of years for the production of alcohol and organic acids and has been the most important metabolic process known to humans. In 1897 this process was opened to scientific scrutiny when Büchner (3) disrupted yeast cells and observed the enzymatic conversion of glucose to ethanol and carbon dioxide. Many studies have addressed the fundamental question of what controls the flux through glycolysis, and much work has focused on analyzing the control by enzymes of the glycolytic pathway. Surprisingly, none of the glycolytic enzymes exerted significant control on the pathway flux in yeast (25) and, in Escherichia coli, overexpression of the proteins that catalyze the uptake and phosphorylation of the glucose did not increase the flux (23).How is the flux through this major metabolic pathway controlled? According to metabolic control theory (9, 19), the sum of control exerted on the glycolytic flux should add up to 1. However, metabolic control theory also postulates that flux control can be shared by many enzymes in a pathway and that control could also reside outside the pathway, for instance, in the processes that consume the ATP generated in glycolysis (the ATP demand). To address the issue of whether ATP consumption by cellular processes determines the steady-state flux through glycolysis, one could augment the existing cellular ATP consumption. However, virtually all ATP-consuming processes are coupled to useful reactions, such as biosynthesis and substrate uptake. Consequently, such a manipulation will affect other processes than ATP consumption.A way to circumvent this problem would be to introduce an ATP-...

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