Ethanologenesis and respiration in a pyruvate decarboxylase-deficient Zymomonas mobilis

Objective Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner–Doudoroff (E–D) glycolysis, pyruvate decarboxylase (Pdc) and two alcohol dehydrogenase (ADH) isoenzymes. Pyruvate is the end-product of the E–D pathway and the substrate for Pdc. Construction and study of Pdc-deficient strains is of key importance for Z. mobilis metabolic engineering, because the pyruvate node represents the central branching point, most novel pathways divert from ethanol synthesis. In the present work, we examined the aerobic metabolism of a strain with partly inactivated Pdc. Results Relative to its parent strain the mutant produced more pyruvate. Yet, it also yielded more acetaldehyde, the product of the Pdc reaction and the substrate for ADH, although the bulk ADH activity was similar in both strains, while the Pdc activity in the mutant was reduced by half. Simulations with the kinetic model of Z. mobilis E-D pathway indicated that, for the observed acetaldehyde to ethanol production ratio in the mutant, the ratio between its respiratory NADH oxidase and ADH activities should be significantly higher, than the measured values. Implications of this finding for the directionality of the ADH isoenzyme operation in vivo and interactions between ADH and Pdc are discussed.

Plasticity of the Pyruvate Node Modulates Hydrogen Peroxide Production and Acid Tolerance in Multiple Oral Streptococci

ABSTRACTCommensalStreptococcus sanguinisandStreptococcus gordoniiare pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H2O2, which is crucial for inhibiting competing biofilm members, especially the cariogenic speciesStreptococcus mutans. H2O2production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H2O2production byS. sanguinisandS. gordonii.S. sanguinisH2O2production was not found to be affected by moderate changes in environmental pH, whereasS. gordoniiH2O2production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H2O2or lactic acid production, revealed increased lactic acid levels forS. gordoniiat pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival ofS. gordoniiat low pH and seems to constitute part of the acid tolerance response ofS. gordonii. Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H2O2.IMPORTANCEOral biofilms are subject to frequent and dramatic changes in pH.S. sanguinisandS. gordoniican compete with caries- and periodontitis-associated pathogens by generating H2O2. Therefore, it is crucial to understand howS. sanguinisandS. gordoniiadapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival, while others maintain their H2O2production at a constant level. The differential control of H2O2production provides important insights into the role of environmental conditions for growth competition of the oral flora.

Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

ABSTRACT 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.