Integrated control of redox and energy metabolism by the membrane-bound and soluble transhydrogenases of Pseudomonas putida across metabolic regimes.

Abstract

Redox homeostasis underpins microbial physiology and stress adaptation, yet the extent to which transhydrogenases contribute to these processes remains largely unexplored in most organisms. In this study, we reveal how Pseudomonas putida integrates two distinct transhydrogenases (membrane-bound PntAB and soluble SthA) into a flexible and reversible redox-balancing system supporting metabolic robustness across diverse conditions. Single deletions of either enzyme had minimal fitness impacts, whereas the double-mutant ΔpntAB ΔsthA exhibited growth defects, disrupted energy charge, and redox imbalance. Specifically, SthA proved essential for growth on acetate via a transcriptional regulator linked to glyoxylate metabolism. Notably, the activity of both transhydrogenases enabled cellular tolerance to formate by channeling the reducing equivalents released from the one-carbon (C₁)-compound oxidation toward NAD^⁺ regeneration. These findings delineate transhydrogenases as dynamic regulators of redox metabolism and energy conservation. Furthermore, this work positions P. putida as a prime host for redox-intensive applications and informs design principles for C₁-based metabolic engineering. A record of this paper's transparent peer review process is included in the supplemental information.