Control of glycolytic flux inZymomonas mobilis by glucose 6-phosphate dehydrogenase activity

Abstract

Glycolytic genes in Zymomonas mobilis are highly expressed and constitute half of the cytoplasmic protein. The first four genes (glf, zwf, edd, glk) in this pathway form an operon encoding a glucose permease, glucose 6‐phosphate dehydrogenase (G6‐P dehydrogenase), 6‐phosphogluconate dehydratase, and glucokinase, respectively. Each gene was overexpressed from a tac promoter to investigate the control of glycolysis during the early stages of batch fermentation when flux (qCO2) is highest. Almost half of flux control appears to reside with G6‐P dehydrogenase (CJG6‐P dehydrogenase = 0.4). Although Z. mobilis exhibits one of the highest rates of glycolysis known, recombinants with elevated G6‐P dehydrogenase had a 10% to 13% higher glycolytic flux than the native organism. A small increase in flux was also observed for recombinants expressing glf. Results obtained did not allow a critical evaluation of glucokinase and this enzyme may also represent an important control point. 6‐Phosphogluconate dehydratase appears to be saturating at native levels. With constructs containing the full operon, growth rate and flux were both reduced, complicating interpretations. However, results obtained were also consistent with G6‐P dehydrogenase as a primary site of control. Flux was 17% higher in operon constructs which exhibited a 17% increase in G6‐P dehydrogenase specific activity, relative to the average of other operon constructs which contain a frameshift mutation in zwf. It is unlikely that all flux control residues solely in G6‐P dehydrogenase (calculated CJG6‐P dehydrogenase = 1.0) although these results further support the importance of this enzyme. As reported in previous studies, changes in flux were not accompanied by changes in growth rate providing further evidence that ATP production does not limit biosynthesis in rich complex medium. © 1996 John Wiley & Sons, Inc.

Acetaldehyde Stimulation of the Growth of Zymomonas mobilis Subjected to Ethanol and Other Environmental Stresses: Effect of Other Metabolic Electron Acceptors and Evidence for a Mechanism

“…The Entner-Doudoroff pathway used by Z. mobilis for catabolism can be affected by changes in the redox balance at both glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Snoep et al [27] found that the flux of glycolysis in Z. mobilis is limited by the activity of glucose-6-phosphate dehydrogenase, both during normal operation of the Entner-Doudoroff pathway (Figure 6) and when the organism is exposed to an excess of ethanol. An increased intracellular concentration of NAD + due to acetaldehyde addition would be predicted to increase glucose-6-phosphate dehydrogenase activity and also augment glyceraldehyde-3-phosphate dehydrogenase activity.…”